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.

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.

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.

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.

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.

Molecular characterization of an estrogen receptor and estrogen-related receptor and their autoregulatory capabilities in two Mytilus species.

Vertebrate-like sex steroid hormones have been widely detected in mollusks, and numerous experiments have shown the importance of steroids in gonad development. Nevertheless, their signaling pathways in invertebrates have not been uncovered yet. Steroid receptors are an ancient class of transcription factors with multiple roles in not only vertebrates but also invertebrates. Estrogen signaling is thought to have major roles in mollusk physiology, but the full repertoire of estrogen receptors is unknown. We presented the successful cloning of two novel forms of estrogen receptor-like genes. These receptors are present in two closely related species of Mytilus: Mytilus edulis and Mytilus galloprovincialis, commonly known and widely distributed sentinel species. Our phylogenetic analysis revealed that one of these receptors is an estrogen receptor (ER) and the other one is an estrogen-related receptor (ERR). Studies of expression analysis showed that both receptor mRNAs were localized in the oocytes and follicle cells in contact with developing oocytes in the ovary and Sertoli cells in the testis, and in the ciliated cells of the gill. In addition, we have evidence that one (ER) of these may have a capacity to autoregulate its own expression in the gonadal cells by estrogen (E2) and that this gene is responsive to estrogenic compounds.

Circadian rhythmicity and photic plasticity of myosin gene transcription in fast skeletal muscle of Atlantic cod (Gadus morhua).

The circadian rhythm is a fundamental adaptive mechanism to the daily environmental changes experienced by many organisms, including fish. Myosins constitute a large family of contractile proteins that are essential functional components of skeletal muscle. They are known to display thermal plasticity but the influence of light on myosin expression remains to be investigated in fish. In the present study, we have examined the circadian rhythmicity and photoperiodic plasticity of myosin gene transcription in Atlantic cod (Gadus morhua) fast skeletal muscle. In silico mining of the Atlantic cod genome resulted in the identification of 76 myosins representing different classes, many of which were hitherto uncharacterized. Among the 23 fast skeletal muscle myosin genes, myh_tc, myh_n1, myh_n4, myo18a_2, and myo18b_2 displayed circadian rhythmic expression and contained several circadian-related transcription factor binding sites (Creb, Mef2 and E-box motifs) within their putative promoter regions. Also, the circadian expression of these 5 myosins strongly correlated with the transcription pattern of clock genes in fast skeletal muscle. Under ex vivo conditions, myosin transcript levels lost their circadian rhythmicity. Nonetheless, different photoperiod regimes influenced the mRNA levels of myh_n4, myo18a_2 and myo18b_2 in fast skeletal muscle explants. Photoperiod manipulation in Atlantic cod juveniles revealed that continuous light significantly elevated mRNA levels of several myosins in fast skeletal muscle when compared to natural photoperiod. The daily rhythmicity observed in some fast skeletal muscle myosin genes suggests that they may be under circadian clock regulation. In addition, the influence of photoperiod on their expression implies that myosins may be involved in the photic plasticity of muscle growth observed in Atlantic cod.

Sexually dimorphic transcription of estrogen receptors in cod gonads throughout a reproductive cycle.

The role of sex steroid regulation in gonadal maturation is a very complex process that is far from being fully understood. Hence, we have investigated seasonal changes in gonadal expression of estrogen receptors (ERs) in Atlantic cod (Gadus morhua L.), a batch spawner, throughout the annual reproductive cycle. Three nuclear ER partial cDNA sequences (esr1, esr2a, and esr2b) were cloned and all esr transcripts were detected mainly in liver and gonads of fish of both sexes. In situ hybridization of esrs along with germ cell (vasa) and gonadal somatic cell markers (gonadal soma-derived factor (gsdf), 3ß-hydroxysteroid dehydrogenase (3ßhsd), and anti-Müllerian hormone (amh) for testicular, or gsdf for ovarian somatic cells) showed that all three esrs were preferentially localized within interstitial fibroblasts composed of immature and mature Leydig cells in testis, whereas they were differentially expressed in both follicular cells and oocytes in ovary. Quantitative real-time PCR analysis revealed a sexually dimorphic expression pattern of the three esr paralogs in testis and ovary. A significant increase in esr2a expression was identified in testis and of esr2b in ovary, whereas esr1 transcripts were elevated in both testis and ovary in February and March before the spawning period. The localization and sexually dimorphic expression of esr genes in gonads indicate a direct function of estrogen via ERs in gonadal somatic cell growth and differentiation for Leydig cell in testis and follicular cells in ovary throughout the annual reproductive cycle in Atlantic cod.

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.

Photoperiod influences growth and mll (mixed-lineage leukaemia) expression in Atlantic cod.

Photoperiod is associated to phenotypic plasticity of somatic growth in several teleost species. However, the molecular mechanisms underlying this phenomenon are currently unknown but it is likely that epigenetic regulation by methyltransferases is involved. The MLL (mixed-lineage leukaemia) family comprises histone methyltransferases that play a critical role in regulating gene expression during early development in mammals. So far, these genes have received scant attention in teleost fish. In the present study, the mean weight of Atlantic cod juveniles reared under continuous illumination was found to be 13% greater than those kept under natural photoperiod conditions for 120 days. We newly determined cDNA sequences of five mll (mll1, mll2, mll3a, mll4b and mll5) and two setd1 (setd1a and setd1ba) paralogues from Atlantic cod. Phylogenetic analysis revealed that the cod genes clustered within the appropriate mll clade and comparative mapping of mll paralogues showed that these genes lie within a region of conserved synteny among teleosts. All mll and setd1 genes were highly expressed in gonads and fast muscle of adult cod, albeit at different levels, and they were differentially regulated with photoperiod in muscle of juvenile fish. Following only one day of exposure to constant light, mll1, mll4b and setd1a were up to 57% lower in these fish compared to the natural photoperiod group. In addition, mRNA expression of myogenic regulatory factors (myog and myf-5) and pax7 in fast muscle was also affected by different photoperiod conditions. Notably, myog was significantly elevated in the continuous illumination group throughout the time course of the experiment. The absence of a day/night cycle is associated with a generalised decrease in mll expression concomitant with an increase in myog transcript levels in fast muscle of Atlantic cod, which may be involved in the observed epigenetic regulation of growth by photoperiod in this species.

Manipulation of fish germ cell: visualization, cryopreservation and transplantation.

Germ-cell transplantation has many applications in biology and animal husbandry, including investigating the complex processes of germ-cell development and differentiation, producing transgenic animals by genetically modifying germline cells, and creating broodstock systems in which a target species can be produced from a surrogate parent. The germ-cell transplantation technique was initially established in chickens using primordial germ cells (PGCs), and was subsequently extended to mice using spermatogonial stem cells. Recently, we developed the first germ-cell transplantation system in lower vertebrates using fish PGCs and spermatogonia. During mammalian germ-cell transplantation, donor spermatogonial stem cells are introduced into the seminiferous tubules of the recipient testes. By contrast, in the fish germ-cell transplantation system, donor cells are microinjected into the peritoneal cavities of newly hatched embryos; this allows the donor germ cells to migrate towards, and subsequently colonize, the recipient genital ridges. The recipient embryos have immature immune systems, so the donor germ cells can survive and even differentiate into mature gametes in their allogeneic gonads, ultimately leading to the production of normal offspring. In addition, implanted spermatogonia can successfully differentiate into sperm and eggs, respectively, in male and female recipients. The results of transplantation studies in fish are improving our understanding of the development of germ-cell systems during vertebrate evolution.