Misako Miwa

Sexual plasticity of ovarian germ cells in rainbow trout

The sexual plasticity of fish gonads declines after the sex differentiation period; however, information about the plasticity of the germ cells themselves after sex differentiation is limited. Using rainbow trout (Oncorhynchus mykiss), we recently established a novel germ cell transplantation system that provides a unique platform with which to dissect the developmental and cellular mechanisms underlying gametogenesis. Using this technique, we show here that transplanted ovarian germ cells isolated from 6- to 9-month-old donors can colonize sexually undifferentiated embryonic gonads and resume gametogenesis. Ovarian germ cells containing oogonia and early oocytes isolated from female rainbow trout were transplanted into the peritoneal cavities of hatching-stage fry of both sexes and the behavior of the donor cells was observed. The transplanted ovarian germ cells migrated towards the recipient gonads, interacted with embryonic gonadal somatic cells, proliferated rapidly, and eventually differentiated into eggs in female recipients and sperm in male recipients. Furthermore, the donor-derived eggs and sperm obtained from the recipient fish were functional and were able to produce normal offspring. These findings indicate that mitotic germ cells, the oogonia, possess a high level of sexual plasticity.

Development of Spermatogonial Cell Transplantation in Nibe Croaker, Nibea mitsukurii (Perciformes, Sciaenidae)

In a recently established system for intraperitoneal spermatogonial cell transplantation in salmonids, donor type A spermatogonia (type A SG) were microinjected into the peritoneal cavity of newly hatched larvae. Compared with salmonids, the larvae of marine teleosts are small and vulnerable to physiological and physical stresses, making it difficult to use them for cell manipulation. Herein, we developed type A SG cell transplantation in Nibe croaker (Nibea mitsukurii) by optimizing 1) the developmental stage of the donor testes used to prepare type A SG-enriched cell suspensions and 2) the timing and location of intraperitoneal cell transplantations to recipient larvae. Donor cells labeled with PKH26 fluorescent dye were transplanted into the peritoneal cavity of 3-, 4-, 5-, and 6-mm larvae using glass micropipettes. Consequently, 20.6% of the 4-mm larvae recipients survived for 3 wk, and 36.3% of the survivors had donor-derived cells in their gonads. The incorporated donor cells were identified as germ cells by germ cell-specific nuclear morphology and expression of a germ cell marker. In contrast, no donor type A SG were incorporated into the gonads of 6-mm recipient larvae. These data indicate that there is a distinct narrow window in the developmental stages of recipient larvae when exogenous type A SG can be incorporated into the gonads. The establishment of this system in pelagic egg-spawning marine teleosts would allow the creation of a new broodstock system in which a target species with a large body size and long generation time could be produced from related species with a small body size and short generation time.

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. Mol. Reprod. Dev.

Characterization of lymphocyte antigen 75 (Ly75/CD205) as a potential cell-surface marker on spermatogonia in Pacific bluefin tuna Thunnus orientalis

In spermatogonial transplantation using Pacific bluefin tuna Thunnus orientalis as a donor, enrichment of spermatogonia (SG) is expected to facilitate high colonization efficiency. Although it is desirable to establish a bluefin tuna SG enrichment procedure using cell-surface markers, a germ cell-specific cell-surface marker has not been identified to date. We previously found that Ly75 is a mitotic germ cell-specific cell-surface marker in rainbow trout, and that its amino-acid sequences are highly conserved in various teleosts. Thus, the ly75 gene is an excellent candidate cell-surface marker of SG in bluefin tuna. In this study, the bluefin tuna ly75 homolog was cloned and characterized for further use as a germ cell-specific cell-surface marker. In adult tissues, high levels of ly75 transcripts were detected in the liver, pyloric caeca, and testis. In situ hybridization analyses showed that ly75 mRNA was predominantly localized in type-A spermatogonia (A-SG), including single A-SG that contain transplantable germ cells. In contrast, ly75 mRNA was not detected in spermatocytes, spermatids, or gonadal somatic cells in testis. The expression profiles of Ly75 protein were similar to those of the mRNA. Therefore, Ly75 is appropriate for use as a cell-surface marker of SG in bluefin tuna.

Expression patterns of gdnf and gfrα1 in rainbow trout testis

In mice, glial cell line-derived neurotrophic factor (GDNF) is essential for normal spermatogenesis and in vitro culture of spermatogonial stem cells. In murine testes, GDNF acts as paracrine factor; Sertoli cells secrete it to a subset of spermatogonial cells expressing its receptor, GDNF family receptor α1 (GFRα1). However, in fish, it is unclear what types of cells express gdnf and gfrα1. In this study, we isolated the rainbow trout orthologues of these genes and analyzed their expression patterns during spermatogenesis. In rainbow trout testes, gdnf and gfrα1 were expressed in almost all type A spermatogonia (ASG). Noticeably, unlike in mice, the expression of gdnf was not observed in Sertoli cells in rainbow trout. During spermatogenesis, the expression levels of these genes changed synchronously; gdnf and gfrα1 showed high expression in ASG and decreased dramatically in subsequent developmental stages. These results suggested that GDNF most likely acts as an autocrine factor in rainbow trout testes.

Combining next‐generation sequencing with microarray for transcriptome analysis in rainbow trout gonads

Microarray technology is a powerful tool for studying genome‐wide gene expression. As the genome of many fish has not yet been determined, however, cDNA microarrays can only be designed from limited expressed sequence tag data. In this study, we designed a microarray based on the sequencing data (337,466 reads) obtained by next‐generation sequencing of RNA extracted from rainbow trout (Oncorhynchus mykiss) embryonic genital ridge, testis, and ovary. These data (307,264 reads) were assembled into 28,668 contigs; 3,298 reads could not be assembled and 26,904 reads were unique sequences that did not cluster with other reads. Based on this information, 55,928 microarray probes were designed for a microarray, which was validated by hybridization experiments with RNA extracted from type A spermatogonia (A‐SG) and testicular somatic cells. Expression of known spermatogonial markers was confirmed to be higher in A‐SG than in testicular somatic cells whereas supporting‐cell markers were expressed at higher levels in testicular somatic cells. This microarray analysis revealed that 8,068 transcripts showed at least fourfold higher signal in A‐SG than testicular somatic cells. Fourteen of 17 randomly selected transcripts were expressed at significantly higher‐levels in A‐SG than somatic cells, by quantitative RT‐PCR. In addition, three transcripts analyzed with in situ hybridization showed A‐SG‐specific signals in immature trout testis, with one of them exhibiting a heterogeneous expression pattern in A‐SG. The rainbow trout gonad microarray developed in this study therefore appears to be a useful tool to understand gametogenesis in rainbow trout. Mol. Reprod. Dev. 79: 870–878, 2012.

Isolation and characterization of a germ cell marker in teleost fish Colossoma macropomum

The native Amazonian fish tambaqui (Colossoma macropomum) is the second-largest scaled fish in South America and the most common native species in Brazil. To preserve genetic resources with sufficient genetic diversity through germ cell cryopreservation and transplantation techniques, a molecular marker for identifying the cells is required to trace them during the manipulation processes. The vasa gene is a promising candidate, as its specific expression in germ cell lineage has been well-conserved throughout animal evolution. In this study, the full sequence of the vasa cDNA homolog from tambaqui was isolated and characterized, showing an open reading frame of 2010 bp encoding 669 amino acids. The putative protein was shown to contain eight conserved motifs of the DEAD-box protein family and high similarity to vasa homologs of other species. Tambaqui vasa (tvasa) mRNA expression was specific to the gonads, and in situ hybridization showed signals only in oocytes and spermatogonia. The results suggested that tvasa could be a useful germ cell marker in this species.