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.     

Characteristics of two populations of Thai river sprat Clupeichthys aesarnensis from man-made reservoirs in Thailand and Laos,with aspects of gonad development

Fisheries Science - Growth and reproduction of the Thai river sprat Clupeichthys aesarnensis (Teleostei: Clupeidae) in Sirindhorn Reservoir, Thailand, and Nam Ngum Reservoir, Laos, were...     

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.     

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.     

Cetacean Toll-like receptor 4 and myeloid differentiation factor 2, and possible cetacean-specific responses against Gram-negative bacteria

Toll-like receptor 4 (TLR4) and myeloid differentiation factor 2 (MD-2) are essential for recognizing the lipopolysaccharides (LPS) of Gram-negative bacteria. We determined the sequences of cDNAs encoding TLR4 and MD-2 from cetaceans and generated three-dimensional (3D) models for a better understanding of their modes of interaction and LPS recognition. The 3D reconstructions showed that cetacean TLR4 and MD-2 formed a horseshoe-like structure comprised of parallel β-strands and a β-cup structure consisting of two anti-parallel β-sheets, respectively. The (TLR4-MD-2)₂ duplex-heterodimer was shown to form a symmetrical structure. Comparison with the interfaces of the complexes in other mammals revealed that cetacean TLR4s have some amino acid residue substitutions involved in duplex-heterodimer formation and in species variation for LPS recognition. These substitutions in the changed amino acid residues may alter the interaction among TLR4, MD-2, and LPS and modify the TLR4/MD-2 immunological responses.     

Aberrant branch of the bronchoesophageal artery resembling patent ductus arteriosus in a dog

An anomalous shunt between the bronchoesophageal artery and pulmonary artery was diagnosed in a 1-year- old, 3.5 kg female Miniature Dachshund by selective contrast angiography. A cardiac murmur had been observed in the dog during examination at another hospital. The machinery murmur was auscultated at the left side of the base of the heart. Although thoracic radiography revealed mild cardiomegaly, the characteristic findings of patent ductus arteriosus (PDA), including as aortic arch enlargement and pulmonary artery enlargement were not observed. Echocardiography demonstrated shunting of blood flow presumably from the arterial duct at the pulmonary artery carina. Based on the above findings the case was diagnosed as PDA. Angiocardiography was performed to confirm the diagnosis in preparation for surgical treatment, but later we confirmed that the shunt vessel was not PDA, but apparently a branch of the bronchoesophageal artery. The shunt vessel was branching in a complicated manner and shunted to the pulmonary artery.     

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?Csinus gland complex (XO?CSG) in the eyestalks. Among the CHH-family peptides analyzed, only the level of sinus gland peptide-G (SGP-G) in the XO?CSG 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?CSG 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.     

Alterations of pattern in immune response and vitellogenesis during induced ovarian development by unilateral and bilateral ablation in Litopenaeus vannamei

Eyestalk ablation is often used to induce ovarian development in female shrimp, but it possibly alters the animal’s hormonal balance, and affects its physiology and immune system. Therefore, this study was conducted to investigate alterations to the immune response during induced ovarian development by unilateral and bilateral ablation in Litopenaeus vannamei. Sub-adult female shrimp could be induced to undergo ovarian development over a 20-day period by both unilateral and bilateral eyestalk ablation; however, ovaries did not reach the final mature stage in which cortical rods are observed. In terms of immune response, total hemocyte count decreased significantly at 4 h after unilateral ablation, and at 20 days after bilateral ablation. After 20 days, expression in the hemocytes of the immune-related genes prophenoloxidase and peroxinectin in bilaterally ablated shrimp increased significantly, whereas that for penaeidin, for lipopolysaccharide- and β-glucan-binding protein, and for transglutaminase mRNA expression did not change significantly. The results of this study suggest that, while eyestalk ablation induces ovarian development, it may also bring about a hormonal imbalance that leads to immune-related genes being activated.     

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.