Gene delivery available in molluscan cells by strong promoter discovered from bivalve-infectious virus.

Understanding gene functions in marine invertebrates has been limited, largely due to the lack of suitable assay systems. Such a system requires investigative methods that are reproducible and can be quantitatively evaluated, such as a cell line, and a strong promoter that can drive high expression of a transgene. In this study, we established primary cell culture from a marine bivalve mollusc, Mizuhopecten yessoensis. Using scallop primary cells, we optimized electroporation conditions for transfection and carried out a luciferase-based promoter activity assay to identify strong promoter sequences that can drive expression of a gene of interest. We evaluated potential promoter sequences from genes of endogenous and exogenous origin and discovered a strong viral promoter derived from a bivalve-infectious virus, ostreid herpesvirus-1 (OsHV-1). This promoter, we termed OsHV-1 promoter, showed 24.7-fold and 16.1-fold higher activity than the cytomegalovirus immediate early (CMV IE) promoter and the endogenous EF1α promoter, the two most commonly used promoters in bivalves so far. Our GFP assays showed that the OsHV-1 promoter is active not only in scallop cells but also in HEK293 cells and zebrafish embryos. The OsHV-1 promoter practically enables functional analysis of marine molluscan genes, which can contribute to unveiling gene-regulatory networks underlying astonishing regeneration, adaptation, reproduction, and aging in marine invertebrates.

Insights into ADAR gene complement, expression patterns, and RNA editing landscape in Chlamys farreri.

Adenosine Deaminases Acting on RNA (ADARs) are evolutionarily conserved enzymes known to convert adenosine to inosine in double-stranded RNAs and participate in host-virus interactions. Conducting a meta-analysis of available transcriptome data, we identified and characterised eight ADAR transcripts in Chlamys farreri, a farmed marine scallop susceptible to Acute viral necrosis virus (AVNV) infections and mortality outbreaks. Accordingly, we identified six ADAR genes in the Zhikong scallop genome, revised previous gene annotations, and traced alternative splicing variants. In detail, each ADAR gene encodes a unique combination of functional domains, always including the Adenosine deaminase domain, RNA binding domains and, in one case, two copies of a Z-DNA binding domain. After phylogenetic analysis, five C. farreri ADARs clustered in the ADAR1 clade along with sequences from diverse animal phyla. Gene expression analysis indicated CF051320 as the most expressed ADAR, especially in the eye and male gonad. The other four ADAR1 genes and one ADAR2 gene exhibited variable expression levels, with CF105370 and CF051320 significantly increasing during early scallop development. ADAR-mediated single-base editing, evaluated across adult C. farreri tissues and developmental stages, was mainly detectable in intergenic regions (83 % and 85 %, respectively). Overall, the expression patterns of the six ADAR genes together with the editing and hyper-editing values computed on scallops RNA-seq samples support the adaptive value of ADAR1-mediated editing, particularly in the pre-settling larval stages.

Hemocytes of Yesso scallop characterized by cytological, molecular marker, and functional analyses.

Bivalve hemocytes have pivotal role as cellular biodefense. However, no information is available for cytological parameters, marker gene and function of the hemocytes in Yesso scallop, a commercially important aquaculture species worldwide. Due to their extremely strong cell aggregation ability, the scallop hemocytes were not able to assess as a single cell so far. In the present study, we established methodologies for studying the hemocytes of Yesso scallop, assessed cell morphology, measured seasonal fluctuation, and analyzed transcriptomes and cellular behavior during the immune response. Our results showed that the Yesso scallop possesses a single type of leukocyte-type hemocytes similar to other bivalve granulocytes circulating at an average of 1 × 107 cells/ml throughout the year. In addition, we identified five molecular marker genes specific to the scallop hemocytes. These hemocyte markers enabled us to precisely detect the hemocyte localization. Using these markers, we confirmed that tissue transplantation can experimentally induce an immune response, leading to the mobilization of circulating hemocytes for encapsulation. This study provides a comprehensive understanding of scallop hemocytes and their role in the cellular biodefense system of bivalves and various methods for cytological analysis.

Loss of Axdnd1 causes sterility due to impaired spermatid differentiation in mice.

Purpose: Spermiogenesis, the process of deformation of sperm head morphology and flagella formation, is a phenomenon unique to sperm. Axonemal dynein light chain proteins are localized to sperm flagella and are known to be involved in sperm motility. Here, we focused on the gene axonemal dynein light chain domain containing 1 (Axdnd1) with the aim to determine the function of its protein product AXDND1. Methods: To elucidate the role of AXDND1 in spermatogenesis, we generated Axdnd1 knockout (KO) mice using the CRISPR/Cas9 system. The generated mice were subjected to fertility tests and analyzed by immunohistochemistry. Result: The Axdnd1 KO mouse exhibited sterility caused by impaired spermiogenesis during the elongation step as well as abnormal nuclear shaping and manchette, which are essential for spermiogenesis. Moreover, AXDND1 showed enriched testicular expression and was localized from the mid-pachytene spermatocytes to the early spermatids. Conclusion: Axdnd1 is essential for spermatogenesis in the mouse testes. These findings improve our understanding of spermiogenesis and related defects. According to a recent report, deleterious heterozygous mutations in AXDND1 were found in non-obstructive azoospermia (NOA) patients. Therefore, Axdnd1 KO mice could be used as a model system for NOA, which will greatly contribute to future NOA treatment studies.

Novel method for mass producing genetically sterile fish from surrogate broodstock via spermatogonial transplantation†.

A stable system for producing sterile domesticated fish is required to prevent genetic contamination to native populations caused by aquaculture escapees. The objective of this study was to develop a system to mass produce stock for aquaculture that is genetically sterile by surrogate broodstock via spermatogonial transplantation (SGTP). We previously discovered that female medaka carrying mutations on the follicle-stimulating hormone receptor (fshr) gene become sterile. In this study, we demonstrated that sterile hybrid recipient females that received spermatogonia isolated from sex-reversed XX males (fshr (-/-)) recovered their fertility and produced only donor-derived fshr (-) X eggs. Natural mating between these females and fshr (-/-) sex-reversed XX males successfully produced large numbers of sterile fshr (-/-) female offspring. In conclusion, we established a new strategy for efficient mass production of sterile fish. This system can be applied to any aquaculture species for which SGTP and methods for producing sterile recipients can be established.

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

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

In vitro and ex vivo models indicate that the molecular clock in fast skeletal muscle of Atlantic cod is not autonomous.

The notion that the circadian rhythm is exclusively regulated by a central clock has been challenged by the discovery of peripheral oscillators. These peripheral clocks are known to have a direct influence on the biological processes in a tissue or cell. In fish, several peripheral clocks respond directly to light, thus raising the hypothesis of autonomous regulation. Several clock genes are expressed with daily rhythmicity in Atlantic cod (Gadus morhua) fast skeletal muscle. In the present study, myosatellite cell culture and short-term cultured fast skeletal muscle explant models were developed and characterized, in order to investigate the autonomy of the clock system in skeletal muscle of Atlantic cod. Myosatellite cells proliferated and differentiated in vitro, as shown by the changes in cellular and myogenic gene markers. The high expression of myogenic differentiation 1 during the early days post-isolation implied the commitment to myogenic lineage and the increasing mRNA levels of proliferating cell nuclear antigen (pcna) indicated the proliferation of the cells in vitro. Transcript levels of myogenic marker genes such as pcna and myogenin increased during 5 days in culture of skeletal muscle explants, indicating that the muscle cells were proliferating and differentiating under ex vivo conditions. Transcript levels of the clock gene aryl hydrocarbon receptor nuclear translocator-like 2 (arntl2) in myosatellite cells showed no daily oscillation regardless of photoperiod manipulation. On the other hand, mRNA levels of the clock gene circadian locomotor output cycles kaput (clock) showed circadian rhythmicity in 5-day-old skeletal muscle explant under different photoperiod regimes. The expression of arntl2, cryptochrome2 (cry2), period 2a (per2a) and nuclear receptor subfamily 1, group D, member 1 was not rhythmic in muscle explants but photoperiod manipulation had a significant effect on mRNA levels of cry2 and per2a. Taken together, the lack of rhythmicity of molecular clocks in vitro and ex vivo indicate that the putative peripheral clock in Atlantic cod fast skeletal muscle is not likely to be autonomous.

Identification and migration of primordial germ cells in Atlantic salmon, Salmo salar: characterization of vasa, dead end, and lymphocyte antigen 75 genes.

No information exists on the identification of primordial germ cells (PGCs) in the super-order Protacanthopterygii, which includes the Salmonidae family and Atlantic salmon (Salmo salar L.), one of the most commercially important aquatic animals worldwide. In order to identify salmon PGCs, we cloned the full-length cDNA of vasa, dead end (dnd), and lymphocyte antigen 75 (ly75/CD205) genes as germ cell marker candidates, and analyzed their expression patterns in both adult and embryonic stages of Atlantic salmon. Semi-quantitative RT-PCR results showed that salmon vasa and dnd were specifically expressed in testis and ovary, and vasa, dnd, and ly75 mRNA were maternally deposited in the egg. vasa mRNA was consistently detected throughout embryogenesis while dnd and ly75 mRNA were gradually degraded during cleavages. In situ analysis revealed the localization of vasa and dnd mRNA and Ly75 protein in PGCs of hatched larvae. Whole-mount in situ hybridization detected vasa mRNA during embryogenesis, showing a distribution pattern somewhat different to that of zebrafish; specifically, at mid-blastula stage, vasa-expressing cells were randomly distributed at the central part of blastodisc, and then they migrated to the presumptive region of embryonic shield. Therefore, the typical vasa localization pattern of four clusters during blastulation, as found in zebrafish, was not present in Atlantic salmon. In addition, salmon PGCs could be specifically labeled with a green fluorescence protein (GFP) using gfp-rt-vasa 3'-UTR RNA microinjection for further applications. These findings may assist in understanding PGC development not only in Atlantic salmon but also in other salmonids.

Short-term in vitro culturing improves transplantability of type A spermatogonia in rainbow trout (Oncorhynchus mykiss).

Continuous production of sperm within the testes is supported by spermatogonial stem cells capable of both self-renewal and the production of numerous differentiated germ cells. We previously demonstrated that a subpopulation of trout type A spermatogonia transplanted into the body cavity of a recipient embryo incorporated into the genital ridge, where they produced functional gametes within the gonads. Various cell-surface proteins could have played a role in the incorporation of spermatogonia into recipient genital ridges. During the preparation of cell suspensions for transplantation in our experimental protocol, however, dissociation of testis by strong proteases was unavoidable. This was problematic as cell-surface proteins may have been at least partially digested by protease activity. In the present study, recovery of spermatogonial surface proteins using short-term culture prior to transplantation was attempted. It was found that spermatogonia cultured in vitro could be harvested by ethylenediaminetetraacetic acid (EDTA) instead of protease treatment. Furthermore, when cultured spermatogonia collected by EDTA treatment were maintained for 24 hr in vitro, they exhibited high adhesiveness. These cultured spermatogonia also possessed higher survival of transplantation compared to spermatogonia newly dispersed by trypsin treatment. These results indicated that spermatogonia possess a reduced ability to migrate toward, adhere to, and/or be incorporated into the recipient genital ridge immediately after protease treatment. Short-term in vitro culturing, however, could allow spermatogonia to recover the surface proteins required for successful incorporation into the recipient genital ridge.

Steroidogenic potential of the gonad during sex differentiation in the rice field frog Hoplobatrachus rugulosus (Anura: Dicroglossidae).

Prior studies demonstrated that gonadal differentiation in the rice field frog, Hoplobatrachus rugulosus, was of an undifferentiated type since all individuals had ovaries at complete metamorphosis. However, the steroidogenic potential of the gonad is still unknown. In this study, H. rugulosus were obtained by stimulating fertilization in the laboratory under natural light and temperature conditions. The gonads were collected and their steroidogenic potential was evaluated by determining the expression level of messenger RNA (mRNA) encoding for cytochrome P450 17-hydroxylase/C17-20 lyase (CYP17) and cytochrome P450 aromatase (CYP19) using quantitative real-time RT-PCR and the localization of CYP17 mRNA in tissues by in situ hybridization. The CYP17 mRNA levels in males at 4-11 weeks postmetamorphosis were higher than in female and intersex gonads. This corresponded to their localization in the gonadal tissues, where CYP17 signals were specifically detected in the Leydig cells of the testis at 5-16 weeks postmetamorphosis but was undetectable in all ovary samples. The CYP19 mRNA levels in females at 4-11 weeks postmetamorphosis was higher than in male and intersex gonads, which corresponded with gonadal development, indicating the potential steroidogenic function of the ovary. Based on the present results, the role of CYP17 and CYP19 mRNA in sex differentiation in H. rugulosus may occur after gonadal sex differentiation and the steroidogenic potential of the gonads exhibited a sexual dimorphic pattern. These results provide a crucial basis for further research on the developmental biology in anuran species.

Expression and functional analyses for estrogen receptor and estrogen related receptor of Yesso scallop, Patinopecten yessoensis.

Estrogen receptors (ERs) were known as estrogen-activated transcription factors and function as major reproduction regulators in vertebrates. The presence of er genes had been reported in Molluscan cephalopods and gastropods. However, they were considered as constitutive activators with unknown biological functions since reporter assays for these ERs did not show a specific response to estrogens. In this study, we tried characterization of ER orthologues from the Yesso scallop, Patinopecten yessoensis, in which estrogens had been proven to be produced in the gonads and involved in the spermatogenesis and vitellogenesis. Identified ER and estrogen related receptor (ERR) of Yesso scallops, designated as py-ER and py-ERR, conserved specific domain structures for a nuclear receptor. Their DNA binding domains showed high similarities to those of vertebrate ER orthologues, while ligand binding domains had low similarities with them. Both the py-er and py-err expression levels decreased in the ovary at the mature stage while py-vitellogenin expression increased in the ovary by quantitative real-time RT-PCR. Also, the py-er and py-err showed higher expressions in the testis than ovary during the developing and mature period, suggesting both genes might function in the spermatogenesis and testis development. The py-ER showed binding affinities to vertebrate estradiol-17ß (E2). However, the intensity was weaker than the vertebrate ER, indicating scallops might exist endogenous estrogens with a different structure. On the other hand, the binding property of py-ERR to E2 was not confirmed in this assay, speculating that py-ERR was a constitutive activator as other vertebrate ERRs. Further, the py-er was localized in the spermatogonia in the testis and in the auxiliary cells in the ovary by in situ hybridization, indicating its potential roles in promoting spermatogenesis and vitellogenesis. Taken together, the present study demonstrated that py-ER was an authentic E2 receptor in the Yesso scallop and might have functions for the spermatogonia proliferation and vitellogenesis, while py-ERR was involved in the reproduction by undiscovered manners.

Gonadal somatic cell-specific transforming growth factor-ß superfamily member in the Yesso scallop reveals gonadal somatic cell distribution during the reproductive phase.

The objective of this study was to identify the gonadal somatic cells in the Yesso scallop using a novel molecular marker. This study is the first to identify the bone morphogenetic protein 2a (Bmp2a) gene as a gonadal somatic cell-specific gene in this bivalve. We performed a transcriptomic survey to identify the transforming growth factor-ß (TGFß) superfamily members that act in Yesso scallop gonad development. BLAST survey, phylogenetic tree, and RT-PCR analyses screened BMP molecules (i.e., bmp2a and bmp10a), which are members of the TGFß superfamily that show gonad-specific expression. Among the BMPs from the Yesso scallop, in situ hybridization accompanied by RNAscope assay identified that bmp2a mRNA was specifically expressed in the gonadal somatic cells localized in the interspace between germ cells. Real-time quantitative PCR (qPCR) analysis revealed that bmp2a mRNA expression increased during the reproductive phase. The relative expression of bmp2a mRNA was lowest at the beginning of the growing stage and peaked at the mature stage in both sexes. These observations indicate that bmp2a-positive gonadal somatic cells support germ cell growth and differentiation during the reproductive phase for both sexes. This study provides new insights into gonadal somatic cell biology in marine invertebrates and we propose that TGFß signaling is necessary for gonad development in bivalves.

The proliferation and migration of immature germ cells in the mussel, Mytilus galloprovincialis: observation of the expression pattern in the M. galloprovincialis vasa-like gene (Myvlg) by in situ hybridization.

In bivalve, the distribution of primordial germ cells can be traced from early embryogenesis to the veliger larva by the expression of the vasa ortholog. However, the distribution of germ cells from metamorphosis to maturation in bivalves has not been examined extensively. In this study, we used in situ hybridization to observe expression of the Mytilus galloprovincialis vasa-like gene (Myvlg). The distribution of germ cells was clarified in immature mussels. We observed germ cells in adult mussels during the non-reproductive and reproductive seasons. Myvlg was specifically expressed in germ cells. Gametogenesis occurs in acini surrounded by connective tissue. Myvlg expression was detected in spermatogonia, spermatocytes, oogonia, and oocytes. In the non-reproductive season, gametes were not observed in the acini, but Myvlg was expressed in germinal stem cells along the acini. The expression intensity in the non-reproductive season, however, was much weaker than that in the reproductive season. Myvlg-positive cells proliferated during the non-reproductive season. In immature mussels, a pair of germ cell clumps was distributed laterally in the connective tissue between the nephric tubules and posterior byssal retractor muscle. Germ cells were also observed along pericardium. When immature mussels grew, a pair of germ cell clumps migrated anteriorly in the connective tissue along the outer epithelium at the dorsal region of the mantle base between the mantle and gill. The number of germ cells increased significantly as the mussels grew. This is the first report to observe the proliferation and migration of germ cells in immature mussels.

Lymphocyte antigen 75 (Ly75/CD205) is a surface marker on mitotic germ cells in rainbow trout.

In mammals, several cell surface molecular markers have been characterized in order to identify the mitotic germ cells. However, little is known in fish about their cell surface antigen. In this study, we identified lymphocyte antigen 75 (Ly75/CD205) as a germ cell-specific cell surface marker by combination expressed sequence tag analysis of purified type A spermatogonia (A-SG) from immature testis, in silico prediction of membrane proteins, and expression studies. The ly75 transcripts were abundant in the testis and gills, and weak signals were detected in the head kidney and brain. In addition, ly75 mRNA was predominantly localized in the primordial germ cells of newly hatched embryos, A-SG in testis, oogonia, and chromatin nucleolus-stage oocytes in the ovary. In contrast, ly75 mRNA was not detected in spermatocytes, spermatids, spermatozoa, vitellogenic oocytes, or gonadal somatic cells from either males or females. The expression profile of Ly75 protein was similar to that of the mRNA. Furthermore, identification of various fish homologs of ly75 confirmed that their amino acid sequences are well conserved. Therefore, Ly75 may be appropriate for use as a versatile cell surface marker for mitotic germ cells in fish.

Molecular identification of steroidogenesis-related genes in scallops and their potential roles in gametogenesis.

Sex steroids are crucial for controlling gametogenesis and germ cell maturation in vertebrates. It has been proposed that Yesso scallop (Mizuhopecten yessoensis) has the same sex steroids as those animals, but the scallop biosynthetic pathway is unclear. In this study, we characterized several steroidogenesis-related genes in M. yessoensis and proposed a putative biosynthetic pathway for sex steroids that is similar to that of vertebrates. Specifically, we identified several steroidogenesis-related gene sequences that encode steroid metabolizing enzymes: StAR-related lipid transfer (START) protein, 17α-hydroxylase, 17,20-lyase (cyp17a), 17ß-hydroxysteroid dehydrogenase (hsd17b), and 3ß-hydroxysteroid dehydrogenase (hsd3b). We sampled adult scallops throughout their reproductive phase to compare their degree of maturation with their intensity of mRNA expression. Semi-quantitative RT-PCR analysis revealed a ubiquitous expression of transcripts for steroid metabolizing enzymes (i.e., star, cyp17a, hsd17b, and hsd3b) in peripheral and gonadal tissues. Real-time PCR analysis revealed a high level of expression of star3 and cyp17a genes in gonadal tissues at the early stage of cell differentiation in scallops. Interestingly, mRNA expression of hsd3b and hsd17b genes showed a synchronous pattern related to degree of gonad maturity. These results indicate that both hsd3b and hsd17b genes are likely involved in steroidogenesis in scallops. We therefore believe that these steroid-metabolizing enzymes allow scallops to endogenously produce sex steroids to regulate reproductive events.

Phenotypic Stability of Sex and Expression of Sex Identification Markers in the Adult Yesso Scallop Mizuhopecten yessoensis throughout the Reproductive Cycle.

The objective of the present study was to analyze the phenotypic stability of sex after sex differentiation in the Yesso scallop, which is a gonochoristic species that has been described as protandrous. So far, no study has investigated in detail the sexual fate of the scallop after completion of sex differentiation, although bivalve species often show annual sex change. In the present study, we performed a tracking experiment to analyze the phenotypic stability of sex in scallops between one and two years of age. We also conducted molecular marker analyses to describe sex differentiation and gonad development. The results of the tracking experiment revealed that all scallops maintained their initial sex phenotype, as identified in the last reproductive period. Using molecular analyses, we characterized my-dmrt2 and my-foxl2 as sex identification markers for the testis and ovary, respectively. We conclude by proposing that the Yesso scallop is a sex-stable bivalve after its initial sex differentiation and that it maintains a sex-stable maturation system throughout its life. The sex-specific molecular markers identified in this study are useful tools to assess the reproductive status of the Yesso scallop.

Erratum: Nagasawa, K. et al. Phenotypic Stability of Sex and Expression of Sex Identification Markers in the Adult Yesso Scallop Mizuhopecten yessoensis throughout the Reproductive Cycle. Animals 2019, 9(5), 277.

In the published article, "Phenotypic Stability of Sex and Expression of Sex Identification Markers in the Adult Yesso Scallop Mizuhopecten yessoensis throughout the Reproductive Cycle. [...].

Substantial Downregulation of Myogenic Transcripts in Skeletal Muscle of Atlantic Cod during the Spawning Period.

Gonadal maturation is an extremely energy consuming process for batch spawners and it is associated with a significant decrease in growth and seasonal deterioration in flesh quality. Our knowledge about the molecular mechanisms linking sexual maturation and muscle growth is still limited. In the present study, we performed RNA-Seq using 454 GS-FLX pyrosequencing in fast skeletal muscle sampled from two-year-old Atlantic cod (Gadus morhua) at representative time points throughout the reproductive cycle (August, March and May). In total, 126,937 good quality reads were obtained, with 546 nucleotide length and 52% GC content on average. RNA-Seq analysis using the CLC Genomics Workbench with the Atlantic cod reference UniGene cDNA data revealed 59,581 (46.9%) uniquely annotated reads. Pairwise comparison for expression levels identified 153 differentially expressed UniGenes between time points. Notably, we found a significant suppression of myh13 and myofibrillar gene isoforms in fast skeletal muscle during the spawning season. This study uncovered a large number of differentially expressed genes that may be influenced by gonadal maturation, thus representing a significant contribution to our limited understanding of the molecular mechanisms regulating muscle wasting and regeneration in batch spawners during their reproductive cycle.

In Vivo Administration of Scallop GnRH-Like Peptide Influences on Gonad Development in the Yesso Scallop, Patinopecten yessoensis.

Existing research on the role of gonadotropin-releasing hormone (GnRH) in bivalve reproduction is inadequate, even though a few bivalve GnRH orthologs have been cloned. The objective of this paper was to elucidate the in vivo effect of GnRH administration in Yesso scallop reproduction. We performed in vivo administration of scallop GnRH (py-GnRH) synthetic peptide into the developing gonad, and analyzed its effect on gonad development for 6 weeks during the reproductive season. The resulting sex ratio in the GnRH administered (GnRH(+)) group might be male biased, whereas the control (GnRH(-)) group had an equal sex ratio throughout the experiment. The gonad index (GI) of males in the GnRH(+) group increased from week 2 to 24.8% at week 6. By contrast the GI of the GnRH(-) group peaked in week 4 at 16.6%. No significant difference was seen in female GI between the GnRH(+) and GnRH(-) groups at any sampling point. Oocyte diameter in the GnRH(+) group remained constant (about 42-45 µm) throughout the experiment, while in the GnRH(-) group it increased from 45 to 68 µm i.e. normal oocyte growth. The number of spermatogonia in the germinal acini of males in the GnRH(+) group increased from week 4 to 6. Hermaphrodites appeared in the GnRH(+) group in weeks 2 and 4. Their gonads contained many apoptotic cells including oocytes. In conclusion, this study suggests that py-GnRH administration could have a potential to accelerate spermatogenesis and cause an inhibitory effect on oocyte growth in scallops.

Characterization of GnRH-like peptides from the nerve ganglia of Yesso scallop, Patinopecten yessoensis.

There is yet no firm experimental evidence that the evolutionary ancient gonadotropin-releasing hormone GnRH (i.e., GnRH1) also acts in invertebrate gametogenesis. The objective of this paper is to characterize candidate invGnRH peptides of Yesso scallop Patinopecten yessoensis (i.e., peptide identification, immunohistochemical localization, and immunoquantification) in order to reveal their bioactive form in bivalves. Using mass spectrometry (MS), we identified two invGnRH (py-GnRH) peptides from the scallop nerve ganglia: a precursor form of py-GnRH peptide (a non-amidated dodecapeptide; py-GnRH12aa-OH) and a mature py-GnRH peptide (an amidated undecapeptide; py-GnRH11aa-NH2). Immunohistochemical staining allowed the localization of both py-GnRH peptides in the neuronal cell bodies and fibers of the cerebral and pedal ganglia (CPG) and the visceral ganglion (VG). We found that the peptides showed a dimorphic distribution pattern. Notably, the broad distribution of mature py-GnRH in neuronal fibers elongating to peripheral organs suggests that it is multi-functional. Time-resolved fluorescent immunoassays (TR-FIA) enabled the quantification of each py-GnRH form in the single CPG or VG tissue obtained from one individual. In addition, we observed greater abundance of mature py-GnRH in VG compared with its level in CPG, suggesting that VG is the main producing organ of mature py-GnRH peptide and that py-GnRH may play a central regulatory role in neurons of scallops. Our study provides evidence, for the first time, for the presence of precursor and mature forms of invGnRH peptides in the nerve ganglia of an invertebrate.