General and Comparative Endocrinology 204 (2014) 71–79

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Molecular characterization and analysis of a putative 5-HT receptor involved in reproduction process of the pearl oyster Pinctada fucata Qi Wang a,b, Maoxian He a,⇑ a CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China b University of Chinese Academy of Sciences, Beijing 100039, China

a r t i c l e

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Article history: Received 12 December 2013 Revised 18 April 2014 Accepted 6 May 2014 Available online 20 May 2014 Keywords: 5-HT receptor Oocyte maturation Spawning Pinctada fucata

a b s t r a c t 5-HT (5-hydroxytryptamine; serotonin) has been linked to a variety of biological roles including gonad maturation and sequential spawning in bivalve molluscs. To gain a better understanding of the effects of 5-HT on developmental regulation in the pearl oyster Pinctada fucata, the isolation, cloning, and expression of the 5-HT receptor was investigated in this study. A full-length cDNA (2541 bp) encoding a putative 5-HT receptor (5-HTpf) of 471 amino acids was isolated from the ovary of the pearl oyster. It shared 71% and 51% homology, respectively, with the Crassostrea gigas 5-HT receptor and the Aplysia californica 5-HT1ap. The 5-HTpf sequence possessed the typical characteristics of seven transmembrane domains and a long third inner loop. Phylogenetic analysis also indicated that 5-HTpf was classified into the 5-HT1 subtype together with other invertebrate 5-HT1 receptors. Quantitative RT-PCR showed that 5-HTpf is widely expressed in all tissues tested, is involved in the gametogenesis cycle, embryonic and larval development stages, and expression is induced by E2 in ovarian tissues. These results suggest that 5-HTpf is involved in the reproductive process, specifically in the induction of oocyte maturation and spawning of P. fucata. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction The biogenic amine serotonin, or 5-hydroxytryptamine (5-HT), is widely distributed in animals. It acts through multiple receptors to modulate many complex behaviors in vertebrates and invertebrates (Gerhardt and Heerikhuizen, 1997; Tierney, 2001). In mammals, 5-HT is involved in learning, anxiety, emotion, sleep, locomotion, reproduction and pain perception (Bordukalo-Niksic et al., 2010; Green and Backus, 1990; Wang et al., 2010; Weiger, 1997). In invertebrates several studies have been performed on the function and location of 5-HT, especially in annelids, arthropods and molluscs (Siniscalchi et al., 2004; Walker, 1984). In molluscs, 5-HT and its receptors are engaged in neuronal functions including feeding (Kawai et al., 2011), circadian rhythm (Levenson et al., 1999), memory (Kandel, 2001), locomotion (Filla et al., 2004), parturition (Fong and Warner, 1995) and development (Panasophonkul et al., 2009). It also plays an important role in

⇑ Corresponding author. Address: Key Laboratory of Marine Bio-resource Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China. Fax: +86 20 84458964. E-mail address: [email protected] (M. He). http://dx.doi.org/10.1016/j.ygcen.2014.05.010 0016-6480/Ó 2014 Elsevier Inc. All rights reserved.

reproduction through biosynthesis and release of active egg-laying peptide precursor proteins, control and initiation of gamete release, and gamete maturation in several molluscan species (Vaca and Alfaro, 2000; Zatylny et al., 2000). Administration of 5HT has been shown to induce oocyte maturation and spawning (Hamida et al., 2004; Tanabe et al., 2010), and to mediate reinitiation of meiosis in prophase-arrested oocytes, as evidenced by germinal vesicle breakdown (GVBD) (Garnerot et al., 2006; Krantic et al., 1992; Krantic and Rivailler, 1996). However, the major function of 5-HT is in the induction of oocyte maturation (Tanabe et al., 2006). The endogenous 5-HT signal is transmitted to the oocyte through 5-HT receptors (Bandivdekar et al., 1991). A number of hormones play key roles in controlling gonad development and secondary sexual characteristics. Testosterone and estradiol both show transient increases during the spawning stage in both sexes of clams (Garnerot et al., 2006). Studies on bivalves have indicated that estrogens potentiate 5-HT-induced spawning which may be mediated through the induction of 5-HT receptor synthesis (Osada et al., 1998, 2004; Wang and Croll, 2006) resulting in oocyte maturation. In mammals, receptors interacting with 5-HT can be classified into seven different subfamilies (5-HT1—5-HT7). All except the

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5-HT3 receptor, which is a ligand-gated ion channel, belong to the superfamily of G-protein-coupled receptors (GPCRs) (Hoyer et al., 2002). In molluscs, several physiological and pharmacological characterizations have been carried out on 5-HT receptors (Barbas et al., 2002; Hamdan et al., 1999). However, the attempts to classify molluscan 5-HT receptors using vertebrate nomenclature have only been partially successful since these receptors appear to have mixed pharmacological and transductional characteristics in some cases. Accordingly, data from molecular studies are very important, since gene structures and deduced amino acid sequences offer a more definitive way to identify and classify invertebrate 5-HT receptors. To date, several 5-HT receptors have been identified by molecular cloning in molluscs, to our knowledge: 5-HTLym and 5-HT2Lym from the pond snail Lymnaea stagnalis, which are presently regarded as 5-HT1-like and 5-HT2 receptors (Gerhardt et al., 1996; Sugamori et al., 1993). Four were characterized in Aplysia californica. Li et al. (1995) cloned two closely-related 5-HT receptors, which were named Ap5-HTB1 and Ap5-HTB2 and could not be classified according to any of the mammalian subtypes. The third receptor, 5-HT1ap, is categorized in the mammalian 5-HT1 subgroup (Angers et al., 1998). 5-HTap2 is the fourth receptor identified in A. californica. It was demonstrated to be similar to 5-HT1ap and to have an identity 68% similar to 5-HTLym (Barbas et al., 2002). A cDNA encoding a putative 5-HT receptor was isolated from the tropical abalone Haliotis rubra, termed 5-HT1ha (Panasophonkul et al., 2009). A putative 5-HT receptor named 5-HTpy was cloned from the Japanese scallop Patinopecten yessoensis (Tanabe et al., 2010). 5-HT receptors were also obtained from the Pacific oyster Crassostrea gigas (Zhang et al., 2012) and Mytilus edulis (Cubero-Leon et al., 2010). Almost all the 5-HT receptors cloned in molluscs are expressed widely in tissues, with 5-HTLym, 5-HT1ap and 5-HTap2 highly expressed in the CNS, while Ap5-HTB1, 5-HT1ha and 5-HTpy showed high expression in the reproductive system. All these findings clearly show that 5-HT receptors mediate a variety of functions in molluscs, but are especially important in reproduction. The pearl oyster Pinctada fucata, a marine bivalve mollusc, cultivated world-wide, has a very high economic value in pearl production in China. However, there are no reports describing the molecular cloning of the 5-HT receptor in this species. To gain insight into the molecular mechanisms of the 5-HT receptors during the reproductive process, we investigated the molecular structure, distribution and induction of expression of a putative 5-HT receptor named 5-HTpf, which was classified into the 5-HT1 subtype.

2. Materials and methods 2.1. Experimental animals and tissue sampling Two-years-old pearl oysters P. fucata were used as the experimental animal. Samples collected at the growing stage of gonad development were obtained from the Marine Biology Station of the Chinese Academy of Sciences at Daya Bay (Shenzhen, Guangdong, PR China) between October 2011 and November 2012. To clone the 5-HT receptor cDNAs, the ovaries were excised, frozen immediately in liquid nitrogen, and then kept at 80 °C until RNA extraction. The ovary, testis, mantle, visceral ganglion, digestive gland, adductor muscle and gill were also dissected and stored in the same manner for analysis of tissue distribution. The gonadal tissues from different stages were dissected and fixed in Bouin’s solution at 4 °C overnight then used to determine gonadal stages. Gametes were obtained by dissecting the gonads then passing through a 100 lm screen to remove large particles of tissue debris. The eggs were fertilized with sperm in filtered seawater containing 0.005–0.006% (v/v) ammonia at temperatures of 25–26 °C, and different stages of embryos or larvae were collected and stored in liquid nitrogen for RNA extraction. 2.2. Reverse transcription, cloning and sequencing The total RNA used for reverse transcription was extracted from frozen tissues using an E.Z.N.A. mollusc RNA kit according to the manufacturer’s protocol (Omega Biotek Inc., Norcross, GA). One microgram of isolated RNA was used to synthesize first-strand cDNA using the ReverTra Ace-first-strand cDNA synthesis kit (Toyobo Co. Ltd., Osaka, Japan). A BD smart race cDNA amplification kit (Clontech, Mountain View, CA) was used to synthesize SMART cDNA. All the primers used for PCR are listed in Table 1. PCR primers were designed using Primer Premier 5.00 (Premier Biosoft International, Palo Alto, CA). First, the fragment obtained from the transcriptome of P. fucata was amplified using a pair of specific primers, 5HTf1 and 5HTr1. PCR was performed under the following conditions: 3 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 30 s at 57 °C, and 1 min at 72 °C with a final extension step of 10 min at 72 °C. The resulting 467 bp fragment was used to generate full-length cDNA by 50 -RACE and 30 -RACE using adaptor primers UPM, NUP and gene-specific primers 5HT50 -1, 5HT50 -2, 5HT30 -1 and 5HT30 -2. Primers 5HTf2 and 5HTr2 were used to validate the open reading frame (ORF). All PCR amplifications were performed as follows: denaturation at 94 °C for 3 min, followed by 35–40 cycles at

Table 1 Primers used for 5-HTpf cloning and expression analysis. No.

Primer name

Usage

Sense/antisense

Primer sequence (50 –30 )

1 2 3 4 5 6 7

5HTf1 5HTr1 5HT50 -1 5HT50 -2 5HT30 -1 5HT30 -2 UPM mixture

RT-PCR RT-PCR 50 RACE Nested 50 RACE 30 RACE Nested 30 RACE 50 and 30 RACE

Sense Antisense Antisense Antisense Sense Sense Sense

NUP 5HTf2 5HTr2 5HTf3 5HTr3 18S-f 18S-r

50 and 30 RACE RT-PCR(ORF) RT-PCR(ORF) Real-time PCR Real-time PCR Real-time PCR Real-time PCR

Sense Sense Antisense Sense Antisense Sense Antisense

50 -GGGCGAAAGCATCCAGAATATAAGT-30 50 -TTTGTTTAGCACTTCTTCGTCGGATGT-30 50 -CACAAGTCCGAACCTAAATACCAG-30 50 -TCCGTGACAGCCAACGATAGA-30 50 -CTGGTATTTAGGTTCGGACTTGTG-30 50 -TCCGACGAAGAAGTGCTAAACA-30 50 -CTAATACGACTCACTATAGGGCAAGCAGT GGTATCAACGCAGAGT-30 50 -CTAATACGACTCACTATAGGGC-30 50 -AAGCAGTGGTATCAACGCAGAGT-30 50 -TCGGGTCACCCTTGGTCG-30 50 -TGAAAGCAAACATACCAGCAATA-30 50 -CTGGTATTTAGGTTCGGACTTGT-30 50 -ATTTGTTTAGCACTTCTTCGTCG-30 50 -CGTTTCAACAAGACGCCAGTAG-30 50 -ACGAAAAAAAGGTTTGAGAGACG-30

8 9 10 11 12 13 14

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94 °C for 30 s, 55–59 °C for 30–40 s and 72 °C for 1–2 min with a further extension of 10 min at 72 °C. All the PCR products were purified using the E.Z.N.A. gel extraction kit (Omega BioTek), subcloned into the pGEM-T Easy Vector (Promega, Madison, WI) and sequenced using an ABI377 autosequencer. 2.3. Tissue distribution of 5-HTpf The tissue expression pattern of 5-HTpf mRNA was analyzed by real-time quantitative PCR. Total RNA was isolated from 7 tissues, including mantle, digestive gland, adductor muscle, gill, visceral ganglion, ovary and testis (n = 3). Quantitative real-time PCR was performed on a Roche LightCycler 480 real-time PCR system using SYBR green master mix for real-time (TOYOBO) according to the manufacturer’s protocol. The primers used are presented in Table 1. The fragments amplified by real-time PCR were all specific and usable. Parallel amplification of an 18S (GenBank Accession No. AY877529.1) reference transcript was performed using 18S-f and 18S-r primers (Table 1). Real-time PCR was performed as follows: denaturation at 94 °C for 1 min, followed by 40 cycles at 94 °C for 15 s, 15 s at 55 °C and 72 °C for 1 min. Accurate amplification of the target amplicon was assessed by constructing a melting curve. The PCR primer efficiency (E) was calculated by the standard curve method: E = 10(1/slope); this value was optimized for each primer pair by generating standard curves from serial dilutions of each positive cDNA control (104–1 mg) to ensure that E ranged from 1.9 to 2.05. Relative expression value was calculated as follows: equations to calculate normalized expression by LightCyclerÒ480 software 1.5 were:

Normalized ratio ¼ ðconc: target=conc: referenceÞsample : ðconc: target=conc: referenceÞcalibrator : After normalized expression data were obtained, the expression level of the target gene in normal tissue was defined as 1, the relative expression value of each sample was then divided by that from the normal tissue. 2.4. Expression pattern of 5-HTpf in embryonic and larval stages Six developmental stages: fertilized egg, embryo at polar body stage, the trochophore, D-shaped larva, umbo larva and metamorphosis stage were collected and stored at 80 °C. Total RNA extraction and quantitative real-time PCR were performed as described above. All samples were analyzed in triplicate. 2.5. Expression pattern of 5-HTpf in gonads during gametogenesis Gonadal tissues were fixed in Bouin’s solution, sliced into 6 lmsections, and processed using routine histological techniques. The sections were stained with hematoxylin/eosin and examined microscopically to classify the stages of gonadal development. cDNAs of 4 stages of ovary and testis (resting stage, growing stage, mature stage and spawning stage) were synthesized to perform real-time PCR as described above. Five replicates of each sample were analyzed. 2.6. Sex steroid injections To evaluate the effect of E2 (17b-estradiol) and MT (methyltestosterone) on the mRNA expression of 5-HTpf, female oysters at the gonadal growing stage were injected with E2; male oysters at the gonadal growing stage were injected with MT. Steroids were purchased from Sigma–Aldrich. E2 and MT were first dissolved in DMSO (dimethyl sulphoxide, purchased from Sigma), and then diluted in modified Herbst’s artificial seawater (ASW). A 100 lL ali-

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quot of aqueous steroid suspension (1 lg/lL) was injected into the adductor muscle of each male or female oyster. The negative control animals were injected with 100 lL of ASW containing 1% DMSO. Eight male and eight female oysters were randomly collected before injection (blank control), then 6, 12 and 24 h after injection, the gonads were dissected and frozen at 80 °C until RNA extraction. Real-time PCR was performed as described previously. 2.7. Data analysis The public database NCBI-BLAST was used to perform homology analysis of the deduced amino acid sequence. Motif analysis of the deduced protein was performed with InterPro Scan on the ExPASy Proteomics Server. Prediction of serine/threonine phosphorylation sites was performed using the NetPhosK 1.0 server online. Multiple sequence alignments of amino acids were performed with ClustalX (1.81). A phylogenetic tree was constructed by the neighbor-joining method using MEGA4. All other sequences were obtained from GenBank and public genome resources. Quantitative data were expressed as mean ± SEM. Significant differences were tested by one-way ANOVA followed by Duncan’s multiple range tests (P < 0.05). All statistical analyses were performed using SPSS 16.0 (IBM, Armonk, NY). 3. Results 3.1. Cloning and sequence analysis of 5-HTpf A full length cDNA encoding a 5-HT receptor named 5-HTpf was isolated from P. fucata (GenBank Accession No. KF954511). The cDNA sequence was 2541 bp in length, including a 296-bp 50 untranslated region (UTR), a 1416-bp open reading frame (ORF) that encoded a putative protein of 471 amino acids with a calculated molecular weight of 53.55 kDa, and an 829-bp 30 -UTR (Fig. 1). It contained seven hydrophobic stretches, putative transmembrane domains as indicated by hydrophobicity analysis of the deduced amino acid sequence. The transmembrane domains were highly conserved between 5-HTpf and other 5-HT1 receptors. The cloned receptor contained 5 potential sites for N-linked glycosylation in the extracellular N-terminal region and 8 potential sites for phosphorylation by protein kinase A or C with 7 sites located in the third cytoplasmic loop. A relatively long third cytoplasmic loop and a short terminal domain (C-terminal tail) were also present in the sequence. An amino acid sequence alignment of 5-HT receptor homologs from different species is shown in Fig. 2. A relatively high level of amino acid sequence identity (41–71%) was found to exist between 5-HTpf and other 5-HT1 subtype receptors, such as 71% identity with pacific oyster C. gigas, 52% with scallop Mizuhopecten yessoensis, 51% with A. californica 5-HT1, 41% with Mus musculus 5-HT1A, and 42% with Homo sapiens 5-HT1A. Moreover, a higher amino acid sequence similarity was observed within the transmembrane domains, such as 81% with C. gigas, 69% with M. yessoensis, and 53% with both M. musculus and H. sapiens, compared to the intracellular and extracellular regions. However, lower identity was indicated between 5-HTpf and other 5-HT subfamily receptors, such as 35% with M. musculus 5-HT4 and 28% with H. sapiens 5-HT6. A phylogenetic tree was generated with 5-HT receptors of various species, and the analysis showed that 5-HTpf was phylogenetically most closely related to C. gigas, which also belongs to the bivalve family, then clustered with A. californica 5-HT receptor 5HT1, and along with other invertebrate and vertebrate 5-HT receptors, these were classified into the 5-HT1 receptor subfamily (Fig. 3). Three other gene models encoding a 5-HT receptor

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Fig. 1. The nucleotide sequence (upper) and deduced amino acid sequence (one letter code, below) of 5-HTpf (Accession No. KF954511). Numerical designations of the nucleotide and amino acid sequences are shown on the left and right respectively. The assigned termination codon is indicated by the asterisk. The putative transmembrane domains are highlighted with gray and numbered from TM I to TM VII. Serines and threonines within a putative sequence for phosphorylation are indicated by triangles. The potential sites for N-linked glycosylation are indicated by circles. The conserved residues for GPCRs are underlined.

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Fig. 2. Multiple alignment of deduced amino acid sequences of 5-HTpf with other 5-HT receptors isolated from Crassostrea gigas (CraGig 5-HT, Accession No. EKC38511.1), Aplysia californica (ApyCal 5-HT1, Accession No. AAC28786.1), Mizuhopecten yessoensis (MizYes 5-HT, Accession No. BAE72141.1), Planorbella trivolvis (PlaTri 5-HT1, Accession No. AAQ95277.1), Procambarus clarkia (ProCla 5-HT1, Accession No. ABX10973.1), Panulirus interruptus (PanInt 5-HT1, Accession No. AAS18607.1), Macrobrachium rosenbergii (MacRos 5-HT1, Accession No. ACB38667.1), Oryzias latipes (OryLat 5-HT1A, Accession No. XP_004072272.1), Danio rerio (DanRer 5-HT1A, Accession No. NP_001116793.1), Mus musculus (MusMus 5-HT1A, Accession No. NP_032334.2), and Homo sapiens (HomSap 5-HT1A, Accession No. NP_000515.2). Alignment was performed using Clustal W. Identical residues are shown in black, dark and light gray represent strongly or weakly conserved similar amino acids. Dashes indicate gaps. Bars indicate seven putative transmembrane domains, 5-HTpf is underlined.

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Fig. 3. Phylogenetic analysis of the 5-HT receptor proteins. The phylogenetic tree was constructed by MEGA.4 using the neighbor-joining method. The bootstrap values from 1000 replicates are given at each branch node. The scale bar indicates 0.1 amino acid replacements per site. Each sequence is denoted by the species from which it was isolated and followed by its 5-HT receptor subtype. Groups of 5-HT receptors in different receptor subtypes were distinguished by brackets. 5-HTpf was marked by blank circle. 5-HT2 like (Gene model ID: pfu_aug1.0_15448.1_18223.t1), 5-HT4 like (pfu_aug1.0_4712.1_37950.t1) and 5-HT7 like(pfu_aug1.0_7369.1_52941.t1) identified in P. fucata draft genome were marked by blank triangle. Abbreviations and the corresponding GenBank accession number of the reference sequences, see Supplementary Table 1 on line.

ortholog were identified in the P. fucata draft genome, and all these genes encode GPCRs. Only transcripts of 5HT4-like sequences were observed in the cDNA library constructed from mantle tissue (Matsumoto et al., 2013). The three gene models clustered

appropriately to different types of 5-HT receptors, as shown in Fig. 3, PinFuc 5-HT2-like, PinFuc 5-HT4-like and PinFuc 5-HT7-like receptors were grouped with the 5-HT2, 5-HT4 and 5-HT7 clusters, respectively.

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3.2. Tissue distribution of 5-HTpf in P. fucata The tissue expression pattern was analyzed by real-time PCR. The efficiency of primers for 5-HTpf was 1.954, and for 18S was 1.956. The analysis showed that 5-HTpf mRNA was expressed in all tissues examined, with a high level of expression in ovary, moderate level in testis, digestive gland and visceral ganglion, and low expression level in mantle, gill and adductor muscle (Fig. 4).

3.3. Expression of 5-HTpf during the developmental and gametogenesis stages of P. fucata The 5-HTpf mRNAs were expressed in all tested embryonic and larval developmental stages. 5-HTpf mRNA was expressed at a moderate level in the fertilized egg, and dropped to a lower level in the other four developmental stages: polar body stage, blastula stage, trochophore stage and D-shaped larva stage, then expression increased to a peak at the umbo larva stage, and then significantly reduced again during metamorphosis of the larva (Fig. 5). In the female gametogenetic cycle, 5-HTpf mRNA was expressed at a moderate level in the three precocious stages, but expressed at a significantly higher level at the spawning stage compared to the other three stages. Although expression showed a rising tendency in the male gametogenetic cycle, there were no significant differences between the spawning stage and the other three stages in the testis (Fig. 6).

Fig. 6. Expression of 5-HTpf mRNA in the gonads (ovary and testis) of P. fucata during the gametogenetic cycle. The mRNA levels were quantified by real-time RTPCR in: (1) RS, resting stage; (2) GS, growing stage; (3) MS, mature stage; (4) SS, spawning stage. 18S (AY877529) was used as the reference gene. Each value represents the mean ± SEM (n = 5). Means not sharing the same superscript are significantly different. (P < 0.05.)

3.4. The effects of E2 and MT injection on 5-HTpf expression As shown in Fig. 7A and B, expression of the 5-HTpf gene in ovarian tissues injected with E2 was significantly higher than in the control or the MT group. 5-HTpf mRNA levels in the ovary increased significantly above control levels 6 h after injection with 1 lg/lL of E2, then fell by 12 and 24 h post-injection (Fig. 7A). The testis showed lower levels of 5-HTpf gene expression 12 h after injection with 1 lg/lL of MT, then rose to a normal level by 24 h after treatment, at which time expression was not significantly different to control levels (Fig. 7B). 4. Discussion

Fig. 4. Expressions of 5-HTpf mRNA in tissues of P. fucata. The mRNA levels were quantified by real-time RT-PCR in: (1) T, testis (at mature stage); (2) O, ovary (at mature stage); (3) M, mantle; (4) VG, visceral ganglion; (5) G, gill; (6) DG, digestive gland and (7) AM, adductor muscle. 18S (AY877529) was used as the reference gene. Each value represents the mean ± SEM (n = 3). Means not sharing the same superscript are significantly different. (P < 0.05.)

Fig. 5. Expression pattern of 5-HTpf mRNA in embryonic and larval stages of P. fucata. The mRNA levels were quantified by real-time RT-PCR in: (1) FE, fertilized egg; (2) PS, polar body stage; (3) BS, blastula stage; (4) TS, trochophore stage; (5) DS, D-shaped larva stage; (6) US, umbo larva stage and (7) ML, metamorphosis of larva. 18S (AY877529) was used as the reference gene. Each value represents the mean ± SEM (n = 3). Means not sharing the same superscript are significantly different. (P < 0.05.)

In molluscs, 5-HT and its receptors are known to be involved in various neuronal functions, but their major effects are exerted in oocyte maturation. The current study provides important new insight into the 5-HT receptor system in the pearl oyster. In the present study, the cDNA sequence of a 5-HT receptor was cloned from the ovary of P. fucata and designated as 5-HTpf. The molecular structure and homology analysis of the 5-HT receptor-deduced amino acid sequences established that 5-HTpf belongs to the 5-HT1 subtype. The 5-HTpf amino acid sequence displays several key features shared by vertebrate and invertebrate 5-HT1 receptors, such as seven hydrophobic transmembrane domains, a relatively long third cytoplasmic loop, and a short fourth inner terminal domain (Albert and Tiberi, 2001; Tierney, 2001). The tripeptide DRY (Asp181-Arg182-Tyr183) in the TM-III domain, and the NPXXY motif (Asn444-Pro445-Tyr448) in TM-VII are believed to be important for receptor activation in members of subfamily 1A of the GPCRs (Bockaert and Pin, 1999). Dendrogram analysis agrees with the previous results suggesting that 5-HTpf together with other invertebrate 5-HT1 receptors is clustered with the vertebrate 5-HT1 subfamily. All these results of analysis indicate that 5-HTpf is therefore clearly associated with the 5-HT1 receptor subfamily. Since molluscan 5-HT1 subtype receptors have previously been investigated for their involvement in regulating reproductive processes such as oocyte maturation, sperm motility and spawning (Siniscalchi et al., 2004; Tanabe et al., 2010), we hypothesized that 5-HTpf may play a similar role in the pearl oyster. mRNA expression of 5-HTpf was found in all tissues examined, which suggests a wide distribution in the body of P. fucata. This finding is in agreement with the results of previous studies on the tropical abalone Haliotis asinine 5-HT1ha (Panasophonkul et al., 2009), the Japanese scallop 5-HTpy (Tanabe et al., 2010) and 5-HT1ap from A. Californica (Angers et al., 1998). Expression of 5-HTpf in the gonads, VG and DG was consistent with previous

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Fig. 7. Levels of mRNA expression for 5-HTpf mRNA synthesis in the gonad of P. fucata after steroid injection. (A) mRNA expression of 5-HTpf after E2 (17b-estradiol) injection. (B) mRNA expression of 5-HTpf after MT (methyltestosterone) injection. 100 ll E2 or MT suspension at a concentration of 1 lg/mL was injected into the adductor muscle of each female or male oyster, respectively. P. fucata were collected randomly and sacrificed before injection, at 6 h, 12 h and 24 h post-injection. NC, negative control. 18S (AY877529) was used as the reference gene. Each value represents the mean ± SEM (n = 8). Means not sharing the same superscript are significantly different. (P < 0.05.)

studies (Garnerot et al., 2006; Matsutani and Nomura, 1986), suggesting that 5-HT may act as a neurotransmitter in P. fucata. The mRNA of 5-HTpf was also expressed widely in embryonic and larval stages, indicating that 5-HTpf may play a crucial role in early embryonic development. The high expression level of 5-HTpf in gonadal tissues provides evidence that the receptor is relevant to the reproductive process of P. fucata. Although 5-HTpf is expressed widely during the gametogenesis cycle both in ovary and testis, the 5-HTpf expression level increased significantly during the spawning stage in the ovary. These results suggest that 5-HTpf may be synthesized during gametogenesis, interact with 5-HT and trigger spawning in the ovary of P. fucata. Although it is expressed at a higher level in testis during the spawning stage, no significant differences emerged from analysis of mRNA expression in the male gonad; this may be partly due to the great variations between individual specimens and the rarity of spawning stage samples. The results of our experiments were in disagreement with data from other species, in which a higher level was found in males than females in bivalve Venus verrucosa and P. yessoensis (Siniscalchi et al., 2004; Tanabe et al., 2010). The differences in the endogenous regulation of the reproductive process between species will need to be investigated in future studies. Moreover, steroids play an important role in bivalve reproduction, and can promote gamete development. Exogenous androgens such as methyltestosterone (MT) have a gonadal masculinization effect while estrogens show a gonadal feminization effect in molluscs (Liu et al., 2008; Wang and Croll, 2006). Injections of estrogen can affect gametogenesis and gametogenesis-related metabolic pathways in invertebrates (Barker and Xu, 1993; Wang and Croll, 2003, 2007). Effects of E2 may be involved in yolk protein formation, and Saido et al. (1992) suggested that a rise in sensitivity to 5-HT occurs during oocyte growth. Expression of the 5-HT receptor in the oocyte membrane was induced by E2 in the Japanese scallop and the oyster C. gigas (Osada et al., 1998; Tanabe et al., 2010), thus suggesting that injection of E2 enhances 5-HT-induced gamete release via induction of 5-HT receptor expression. In this study, expression of 5-HTpf in ovarian tissues was significantly upregulated 6 h after injection with E2, a finding which supported Osada’s study (1998). Our results suggest that E2 can upregulate 5-HTpf expression in oocytes during oocyte maturation in P. fucata. The possible roles of E2 in the control of reproduction and 5-HTinduced gamete release in molluscs is not established yet, much more work is required to understand mechanisms underlying the actions of E2. Meanwhile, the influence of MT on 5-HTpf in the testis was also investigated, and we found that there were no significant changes of 5-HTpf expression after MT injection. Wang and Croll (2003, 2006) found that testosterone had facilitatory effects on sperm release induced by 5-HT and potentiated spawning in males

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