Kazue Nagasawa

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 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.

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.

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. Mol. Reprod. Dev. 80: 763–773, 2013.

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.

Transgene manipulation in rainbow trout using Cre recombinase

Cre/loxP-mediated cell targeting is considered to be a powerful tool for biotechnology in farmed fish. As a first step toward establishing cell targeting in salmonids, we analyzed the functionality of the Cre/loxP system in rainbow trout. We first established stable transgenic strains carrying the DsRed gene, which was flanked by loxP sites and further spliced with the EGFP gene. By microinjecting Cre complementary RNA (cRNA) into fertilized eggs of the transgenic trout, the functionality of the Cre/loxP system was evaluated. The results showed that all of the embryos exhibited green fluorescence in at least some of their cells. While 19 out of 20 embryos comprised cells showing both green and red fluorescence, the remaining embryo showed only green fluorescence. Polymerase chain reaction (PCR) using primers designed to recognize sequences outside of the two loxP sites revealed that, in addition to long intact fragments, the 19 individuals carried short fragments that were equivalent in length to the loxP-excised fragments. The remaining green embryo carried only this short fragment. DNA sequencing of the short fragment revealed that it lacked the DNA fragments flanking the loxP sites and the spliced fragments did not contain any sequence rearrangements. These results suggest that the Cre/loxP system is functional in rainbow trout.

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), 17β-hydroxysteroid dehydrogenase (hsd17b), and 3β-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.

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

Abstract 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 donorderived 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. Summary Sentence Novel method for mass producing genetically sterile fish from surrogate broodstock was established via transplantation of spermatogonia isolated from follicle-stimulating hormone receptor mutants

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.