Abstracts of the presentations selected for 2004 JSAR Outstanding Presentation Award

Abstracts of the presentations selected for 2005
JSAR Outstanding Presentation Award

Production of Salmon that Generates Only Trout Offspring: Use of Sterilized Triploid as a Recipient

Tomoyuki OKUTSU¹, Goro YOSHIZAKI^1,2 and Toshio TAKEUCHI¹

¹Laboratory of Fish Physiology, Tokyo University of Marine Science and Technology, Minato-ku, Tokyo, Japan; and ²PRESTO, Japan Science and Technology Corporation, Kawaguchi-shi, Saitama, Japan

ABSTRACT. Recently, we described a method to transplant germ cells between two different salmonid species, with the subsequent production of xenogenic, donor-derived offspring. This system is applicable for surrogate broodstock technology and conservation of endangered species. In this system, however, transplanted recipients produce not only donor-derived but also recipient-derived gametes, causing low production rate of donor-derived offspring. In this study, we examined the feasibility of using infertile triploid salmon as a recipient to increase the contribution efficiency of donor-derived gametes. Donor cells were prepared from transgenic male rainbow trout (Onchorhynchus mykiss) (11 mo of age) carrying a green fluorescent protein (GFP) gene driven by the vasa gene regulatory regions. Spermatogonia in the testis of this transgenic trout were labeled by indelible green fluorescence. Approximately 15,000 testicular cells containing 6,000-10,000 germ cells were transplanted into the peritoneal cavity of newly hatched triploid masu salmon (O. masou). Two years after the transplantation, milts were obtained from 10 out of 29 (34.5%) triploid salmon recipients, whereas control triploids without receiving trout germ cells did not show any sign of spermiation. PCR analysis with GFP-specific primers revealed that donor-derived GFP genes were detected in the milts from these 10 recipients. The milts were subsequently used for artificial insemination with non-transgenic trout eggs. All embryos hatched by 35 days post-fertilization (dpf), which is the same timing as that of control trout. On the other hand, F1 hybrid between trout and salmon hatched around 40 dpf. The triploid-recipient-derived hatchlings had GFP-labeled germ cells and grew up normally. Further, randomly amplified polymorphic DNA analysis of the hatchlings suggested that the DNA fingerprint of the all offspring (50 offspring examined) derived from the triploid recipients was identical to that of trout and no DNA fragment derived from salmon was detected. Taken these results together, we concluded that 100% of offspring yielded by the triploid salmon recipients was genuine rainbow trout and no hybrids appeared among them. Thus, triploid is a suitable recipient for efficient production of donor-derived offspring.

PGF_2α Acutely Stimulates the Expression of Nitric Oxide Synthase in Endothelial Cells Cultured with Luteinized Granulosa Cells and Smooth Muscle Cells: A Possible Role of Nitric Oxide in the Increase of Blood Flow Surrounding the Corpus Luteum as a Starting Signal of Luteolysis

Koumei SHIRASUNA, Dai YAMAMOTO, Keiko MOROTA and Akio MIYAMOTO

Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan

ABSTRACT. We previously indicated that the luteal blood flow surrounding the corpus luteum (CL) drastically increases prior to decrease of plasma progesterone (P) in the cow. Furthermore, we suggested that this acute increase of luteal blood flow is a starting signal of luteolysis in response to PGF_2α that may be induced by nitric oxide (NO) as a strong vasorelaxant. The present study therefore investigated the effect of PGF_2α and NO on the mRNA expression of endothelial NO synthase (eNOS) and P secretion by co-culture model. To mimic the environment of local luteal region, the mix-culture of luteinized granulosa cells (LGC; 6 days cultured), bovine aorta endothelial cells (EC) and bovine aorta smooth muscle cells (SMC) was considered as a model for the periphery of CL. In addition, the co-culture of LGC and EC was considered as a model for the inside of CL, or EC and SMC for ovarian artery. After 2-3 days culture, each model was treated with PGF_2α (10^-6 M) or NO donor (S-nitroso-N-acetyl-L-penicillamine; 10^-4 M). At 0, 0.5 and 1 h after treatment, eNOS mRNA was determined by real-time RT-PCR, and P concentrations in medium was measured by EIA. In the model for inside of CL, PGF_2α stimulated eNOS mRNA expression at 1 h. However, in the model for periphery of CL, PGF_2α increased eNOS mRNA at 0.5 h quicker than that in the model for inside of CL. In contrast, PGF_2α had no effect in the model for ovarian artery. NO donor inhibited P secretion from 0.5 h in both inside and periphery of CL models. NO donor suppressed P secretion to 62% in the model for periphery of CL and 44% in the model for inside of CL. The overall results indicate that PGF_2α can stimulate eNOS expression in EC neighboring to LGC but not in EC alone, and this response becomes more rapid due to participation of SMC. Moreover, the effect of NO (NO donor) to inhibit P secretion is stronger in the model for inside of CL than that in the model for periphery of CL. Thus, NO stimulated by PGF_2α may relate to the increase of blood flow surrounding the CL and the decrease of P secretion, suggesting that NO has a crucial role to initiate luteolysis in the cow.

[ Back ]

Production of Salmon that Generates Only Trout Offspring: Use of Sterilized Triploid as a Recipient
Tomoyuki OKUTSU¹, Goro YOSHIZAKI^1,2 and Toshio TAKEUCHI¹
¹Laboratory of Fish Physiology, Tokyo University of Marine Science and Technology, Minato-ku, Tokyo, Japan; and ²PRESTO, Japan Science and Technology Corporation, Kawaguchi-shi, Saitama, Japan

	ABSTRACT. Recently, we described a method to transplant germ cells between two different salmonid species, with the subsequent production of xenogenic, donor-derived offspring. This system is applicable for surrogate broodstock technology and conservation of endangered species. In this system, however, transplanted recipients produce not only donor-derived but also recipient-derived gametes, causing low production rate of donor-derived offspring. In this study, we examined the feasibility of using infertile triploid salmon as a recipient to increase the contribution efficiency of donor-derived gametes. Donor cells were prepared from transgenic male rainbow trout (Onchorhynchus mykiss) (11 mo of age) carrying a green fluorescent protein (GFP) gene driven by the vasa gene regulatory regions. Spermatogonia in the testis of this transgenic trout were labeled by indelible green fluorescence. Approximately 15,000 testicular cells containing 6,000-10,000 germ cells were transplanted into the peritoneal cavity of newly hatched triploid masu salmon (O. masou). Two years after the transplantation, milts were obtained from 10 out of 29 (34.5%) triploid salmon recipients, whereas control triploids without receiving trout germ cells did not show any sign of spermiation. PCR analysis with GFP-specific primers revealed that donor-derived GFP genes were detected in the milts from these 10 recipients. The milts were subsequently used for artificial insemination with non-transgenic trout eggs. All embryos hatched by 35 days post-fertilization (dpf), which is the same timing as that of control trout. On the other hand, F1 hybrid between trout and salmon hatched around 40 dpf. The triploid-recipient-derived hatchlings had GFP-labeled germ cells and grew up normally. Further, randomly amplified polymorphic DNA analysis of the hatchlings suggested that the DNA fingerprint of the all offspring (50 offspring examined) derived from the triploid recipients was identical to that of trout and no DNA fragment derived from salmon was detected. Taken these results together, we concluded that 100% of offspring yielded by the triploid salmon recipients was genuine rainbow trout and no hybrids appeared among them. Thus, triploid is a suitable recipient for efficient production of donor-derived offspring.

PGF_2α Acutely Stimulates the Expression of Nitric Oxide Synthase in Endothelial Cells Cultured with Luteinized Granulosa Cells and Smooth Muscle Cells: A Possible Role of Nitric Oxide in the Increase of Blood Flow Surrounding the Corpus Luteum as a Starting Signal of Luteolysis
Koumei SHIRASUNA, Dai YAMAMOTO, Keiko MOROTA and Akio MIYAMOTO
Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan

	ABSTRACT. We previously indicated that the luteal blood flow surrounding the corpus luteum (CL) drastically increases prior to decrease of plasma progesterone (P) in the cow. Furthermore, we suggested that this acute increase of luteal blood flow is a starting signal of luteolysis in response to PGF_2α that may be induced by nitric oxide (NO) as a strong vasorelaxant. The present study therefore investigated the effect of PGF_2α and NO on the mRNA expression of endothelial NO synthase (eNOS) and P secretion by co-culture model. To mimic the environment of local luteal region, the mix-culture of luteinized granulosa cells (LGC; 6 days cultured), bovine aorta endothelial cells (EC) and bovine aorta smooth muscle cells (SMC) was considered as a model for the periphery of CL. In addition, the co-culture of LGC and EC was considered as a model for the inside of CL, or EC and SMC for ovarian artery. After 2-3 days culture, each model was treated with PGF_2α (10^-6 M) or NO donor (S-nitroso-N-acetyl-L-penicillamine; 10^-4 M). At 0, 0.5 and 1 h after treatment, eNOS mRNA was determined by real-time RT-PCR, and P concentrations in medium was measured by EIA. In the model for inside of CL, PGF_2α stimulated eNOS mRNA expression at 1 h. However, in the model for periphery of CL, PGF_2α increased eNOS mRNA at 0.5 h quicker than that in the model for inside of CL. In contrast, PGF_2α had no effect in the model for ovarian artery. NO donor inhibited P secretion from 0.5 h in both inside and periphery of CL models. NO donor suppressed P secretion to 62% in the model for periphery of CL and 44% in the model for inside of CL. The overall results indicate that PGF_2α can stimulate eNOS expression in EC neighboring to LGC but not in EC alone, and this response becomes more rapid due to participation of SMC. Moreover, the effect of NO (NO donor) to inhibit P secretion is stronger in the model for inside of CL than that in the model for periphery of CL. Thus, NO stimulated by PGF_2α may relate to the increase of blood flow surrounding the CL and the decrease of P secretion, suggesting that NO has a crucial role to initiate luteolysis in the cow.