Tomoyuki Okutsu

Production of Trout Offspring from Triploid Salmon Parents

Many salmonids have become at risk of extinction. For teleosts whose eggs cannot be cryopreserved, developing techniques other than egg cryopreservation to save genetic resources is imperative. In this study, spermatogonia from rainbow trout were intraperitoneally transplanted into newly hatched sterile triploid masu salmon. Transplanted trout spermatogonia underwent spermatogenesis and oogenesis in male and female recipients, respectively. At 2 years after transplantation, triploid salmon recipients only produced trout sperm and eggs. With use of these salmon as parents, we successfully produced only donor-derived trout offspring. Thus, by transplanting cryopreserved spermatogonia into sterile xenogeneic recipients, we can generate individuals of a threatened species.

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.

Spermatogonial transplantation in fish: A novel method for the preservation of genetic resources

Recent progress in genome-based breeding has created various fish strains carrying desirable genetic traits; however, methods for the long-term preservation of their genetic resources have not yet been developed, mainly due to the lack of cryopreservation techniques for fish eggs and embryos. Recently, we established an alternative cryopreservation technique for fish spermatogonia using a slow-freezing method. Furthermore, we developed a transplantation system to produce functional eggs and sperm derived from spermatogonia. Spermatogonia isolated from the testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout (Oncorhynchus mykiss) were transplanted into the peritoneal cavity of triploid masu salmon (Oncorhynchus masou) hatchlings of both genders. The transplanted trout spermatogonia migrated towards the gonadal anlagen of the recipient salmon, into which they were subsequently incorporated. We confirmed that the donor-derived spermatogonia resumed gametogenesis, and produced sperm and eggs in male and female recipient salmon, respectively. Fertilization of the resultant eggs and sperm produced only rainbow trout in the first filial (F₁) generation, suggesting that the sterile triploid recipient salmon produced functional eggs and sperm derived from the trout donors. A combination of spermatogonial transplantation and cryopreservation could be a powerful tool for preserving valuable fish strains with desirable genetic traits and endangered species.

Identification of Novel Genes Associated with Molecular Sex Differentiation in the Embryonic Gonads of Rainbow Trout (Oncorhynchus mykiss)

The molecular pathways in embryonic vertebrates leading to gonad formation in each sex are incompletely understood. The purpose of this study was to identify novel genes that could be associated with sex-specific gonadal differentiation in a fish, the rainbow trout (Oncorhynchus mykiss). This study was facilitated by a custom microarray based on 7,671 genes derived from embryonic rainbow trout gonad cDNA libraries and public databases. Gonad samples for total RNA isolation were obtained from pvasa-green fluorescent protein (pvasa-GFP) transgenic rainbow between 300 and 700 degree-days of development post-fertilization. The transgenic fish permitted the collection of gonads from embryonic rainbow trout during the period of molecular sex differentiation in advance of any morphologically distinguishable characteristics of sex. A bioinformatic method was used with the microarray data that looked for strong associations in gene expression patterns between known sex differentiation genes (the target genes) and novel genes (the target-associated genes) previously not allied with sex differentiation in fishes. The expression patterns of representative target genes from both sexes and their target-associated genes were independently confirmed by real-time reverse transcription polymerase chain reaction to support the validity of the bioinformatic method employed. Numerous novel genes were identified in the gonads of embryonic female and male rainbow trout that could be involved in sex-specific differentiation pathways in this fish.

Biological characteristics of fish germ cells and their application to developmental biotechnology.

We have revealed several unique characteristics of germ cell development using rainbow trout, including the fact that spermatogonia transplanted into the peritoneal cavity of newly hatched embryos migrate toward recipient gonads, that spermatogonia transplanted into female recipients start oogenesis and produce functional eggs and that diploid germ cells transplanted into triploid trout can complete gametogenesis. By combining these unique features of fish germ cells, we established allogeneic and xenogeneic transplantation systems for spermatogonia in several fish species. Spermatogonia isolated from the mature testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout were transplanted into the peritoneal cavity of triploid masu salmon newly hatched embryos. These spermatogonia migrated toward recipient salmon genital ridges with extending pseudopodia and were subsequently incorporated into them. We further confirmed that the donor-derived spermatogonia resumed gametogenesis and produced sperm and eggs in male and female salmon recipients, respectively. By inseminating the resulting eggs and sperm, we obtained only rainbow trout offspring in the F1 generation, suggesting that the triploid salmon recipients produced functional gametes derived only from donor trout. We further confirmed that this intra-peritoneal transplantation of germ cells is applicable to several marine fishes, which could be of benefit in the production of bluefin tuna that has a large broodstock (>100 kg) and is difficult to maintain in captivity. Gamete production of bluefin tuna could be more easily achieved by generating a surrogate species, such as mackerel, that can produce tuna gametes.

Metabolism of amino acids during hyposmotic adaptation in the whiteleg shrimp, Litopenaeus vannamei

The penaeid prawn, Litopenaeus vannamei, was employed to investigate intracellular isosmotic regulation in situations where invertebrates encounter hyposmosis. Hemolymph osmolality was first analyzed to confirm osmoregulatory conditions in the experimental animals, followed by analysis of amino acids in muscle and hemolymph using high-performance liquid chromatography. Total muscle amino acid levels decreased when hemolymph osmolality was extremely low, whereas glycine and l-serine levels increased in the hemolymph. These results suggest that tissue amino acids were released into the hemolymph to lower the osmolality of the tissues for purposes of low-salinity adaptation. Next, oxygen consumption and ammonia excretion rates were examined, and the O/N ratio was determined. Oxygen consumption levels and ammonia excretion rates increased, and the O/N ratio decreased when the animals were exposed to low salinity. These results suggest that amino acids were abundantly consumed as an energy source when animals were exposed to low salinity. To confirm the consumption of particular amino acids, the specific activity of l-serine ammonia lyase was also examined. Specific activity was highest when l-serine levels in the hemolymph were highest. Thus, it appears that l-serine levels increased under hyposmotic conditions due to the consumption of l-serine as an energy source. It was concluded that particular amino acids as osmolytes are likely metabolized as energy sources and consumed for purposes of hyposmotic adaptation.

Production of germ cell-deficient salmonids by dead end gene knockdown, and their use as recipients for germ cell transplantation

We previously established a spermatogonial transplantation model in fish using triploid recipients. Although triploid salmonids are sterile, they carry a limited number of immature triploid germ cells that potentially compete with the donor‐derived germ cells for their niche. We therefore assessed the biological characteristics of germ cell‐deficient gonads in rainbow trout for their suitability as recipients for germ cell transplantation in this study. Antisense morpholino oligonucleotides against the dead end gene were microinjected into the fertilized eggs of rainbow trout to eliminate endogenous germ cells, leaving only their supporting cells. Unlike similar approaches performed in zebrafish and medaka, these germ cell‐deficient rainbow trout did not show a male‐biased sex ratio. Approximately 30,000 spermatogonia were then transplanted into the body cavities of both germ cell‐deficient and control recipients. The donor‐derived germ cells showed significantly higher proliferation in the gonads of germ cell‐deficient recipients than those in the gonads of the control recipients. Finally, the applicability of the germ cell‐deficient recipients for xenogeneic transplantation was evaluated by transplanting rainbow trout spermatogonia into germ cell‐deficient masu salmon recipients. The resulting recipient salmon matured normally and produced trout gametes, and early survival of the resulting trout offspring was as high as that of the control offspring. Thus, dead end‐knockdown salmonids appear to be ideal recipients for the intraperitoneal transplantation of spermatogonia. Mol. Reprod. Dev. 83: 298–311, 2016.

Dynamics of Vitellogenin and Vitellogenesis-Inhibiting Hormone Levels in Adult and Subadult Whiteleg Shrimp, Litopenaeus vannamei: Relation to Molting and Eyestalk Ablation

ABSTRACT Levels of vitellogenin (VG) and vitellogenesis-inhibiting hormone (VIH) in the whiteleg shrimp, Litopenaeus vannamei, were measured by time-resolved fluoroimmunoassay in relation to the molting cycle and ovarian maturation induced by eyestalk ablation. During the molt cycle, VG mRNA expression levels and VG concentrations showed similar patterns of fluctuation. VG levels increased significantly at early intermolt (stage C0) in adults, but not in subadults. Unilateral and bilateral eyestalk ablation increased VG levels in adults, whereas only bilateral eyestalk ablation affected subadults. VIH levels showed contrasting patterns between adults and subadults. In adults, levels were high in late postmolt adults (stage B) and then low thereafter, whereas they increased from postmolt (stage A) to intermolt (stage C0) in subadults and remained high. Unilateral eyestalk ablation increased VIH levels 10 days following ablation in adults, after which levels decreased at 20 days. VIH levels decreased from 10 to 20 days after bilateral ablation. Both unilateral and bilateral ablation led to increased VIH levels in subadults. Eyestalk ablation induced ovarian maturation, but did not reduce VIH concentrations in the hemolymph. This phenomenon was perhaps due to other crustacean hyperglycemic hormone peptides having cross-reactivity with VIH antibodies. This is the first report to quantify concentrations of VG and VIH together in L. vannamei hemolymph, and to examine their relative dynamics.

Molecular cloning of a cDNA encoding vitellogenesis-inhibiting hormone in the whiteleg shrimp Litopenaeus vannamei and preparation of its recombinant peptide using an E. coli expression system

The cDNA encoding a precursor of Liv-SGP-G, the most abundant crustacean hyperglycemic hormone-family peptide in the sinus gland of the whiteleg shrimp Litopenaeus vannamei, was cloned by RT-PCR coupled with 5′- and 3′-RACE. The determined cDNA sequence consisted of 621 nucleotides comprising a 69-bp 5′-untranslated region, a 357-bp open reading frame, and a 195-bp 3′-untranslated region. The deduced sequence of 72 amino acid residues corresponding to mature Liv-SGP-G was completely identical to that of natural Liv-SGP-G, determined in our previous study. The recombinant Liv-SGP-G was thereafter heterologously expressed in Escherichia coli, and its vitellogenesis-inhibiting activity in ex vivo cultured ovarian fragments was examined. The recombinant peptides inhibited vitellogenin gene expression with almost the same efficacy as that of natural Liv-SGP-G purified from sinus glands.

Changes in crustacean hyperglycemic hormones in Pacific whiteleg shrimp Litopenaeus vannamei subjected to air-exposure and low-salinity stresses

Changes in crustacean hyperglycemic hormone (CHH)-family peptides in response to stress were investigated in Litopenaeus vannamei. Stress treatments consisted of air exposure and low salinity. High-performance liquid chromatography was used to quantify CHH-family peptides in the X-organ–sinus gland complex (XO–SG) in the eyestalks. Among the CHH-family peptides analyzed, only the level of sinus gland peptide-G (SGP-G) in the XO–SG was decreased. SGP-G was also detectable by Western blotting analysis in the hemolymph of animals subjected to stress. These results suggest that SGP-G was secreted from the XO–SG into the hemolymph during stress. Glucose levels in the hemolymph increased under conditions during which SGP-G was detected in the hemolymph. Hyperglycemia was also observed when SGP-G was injected. SGP-G may function to shift energy use to deal with stress.