146 LECTIN DOLICHOS BIFLOURUS AGGLUTININ (DBA) IS A SPECIFIC MARKER for PRIMITIVE GERM CELLS IN NEONATAL PORCINE TESTIS

Abstract

Livestock transgenesis is primarily directed towards improving productive traits or for biomedical research. Transgenic pig is of immense interest because it could provide tissues and organs for xenotransplantation to humans. Conventional methods like pronuclear microinjection of zygote and, more recently, somatic cell nuclear transfer (SCNT) have been used for generating genetically modified pigs. These methods are, however, inefficient and have numerous shortcomings. Male germline stem cells have enormous potential, because they could serve as an alternative approach to generate genetically modified pigs, in which the homologous recombination technique could be applied. However, this could not be achieved due to lack of knowledge of culture conditions and markers that can distinguish germ cells from somatic cells, enabling their isolation and identification in culture. Lectin dolichos biflourus agglutinin (DBA), which has specific affinity for ?-d-N-acetyl-galactosamine, reacts with the large round primordial germ cells of the pig genital ridge (Takagi et al. 1997 Mol. Reprod. Dev. 46, 567–580). Immunohistochemical analysis of testicular samples from different age groups revealed that DBA binds to primitive germ cells in neonatal pig testis like gonocytes and pre-spermatogonia. Variable DBA affinity among germ cells and its progressive loss with age suggested that DBA bound strongly to gonocytes, weakly to undifferentiated spermatogonia, and not at all to spermatogonia. The presence of alkaline phosphatase activity in germ cells from neonatal pig testis further confirmed the existence of primitive germ cells. Gonocytes from neonatal pig testis were isolated using 2-step enzymatic digestion and discontinuous Percoll density gradient. Approximately 70% gonocytes were present in the fraction 5 (50–60% gradient) that were identified by their DBA affinity. Isolated cells were cultured in DMEM/F12 supplemented with 10 µg mL^-1 of insulin, 10 µg mL^-1 of apo-transferrin, 100 U mL^-1 of penicillin, 50 µg mL^-1 of streptomycin, 40 µg mL^-1 of gentamycin sulphate, 1 mM pyruvate, 1× non-essential amino acid solution, and 10% fetal bovine serum at 37°C in 5% CO₂ in air in a humidified atmosphere. Initially, gonocytes grew as adherent cells that formed foci of flat colonies. These colonies grew in size during the culture period, and DBA-positive cells were observed. Flat colonies transformed to ES-like colonies by 6–7 days of culture, which were still positive for DBA binding. Proliferation of germ cells was evaluated by double immunostaining with DBA and anti-bromodeoxyuridine (BrdU) antibody following BrdU incorporation, suggesting that 53–55% DBA-positive cells were in S phase after 7 days of culture. In conclusion, lectin DBA is a marker for porcine primitive germ cells in neonatal pig testis including gonocytes that could be used for their identification during isolation and in culture. Isolated gonocytes could proliferate in vitro without specific growth factor supplementation.