Isolation and characterization of rooster (Gallus gallus) spermatogonia

Using the male gonad cells of farm animals and poultry in the biotechnological programs to produce chimeric and genetically modified animals is regarded as an alternative to traditional methods of breeding and genome modification and offers great opportunities for obtaining individuals with new desired properties. In transgenesis, this approach provides a targeted genetic modification of male germ cells by introducing the recombinant DNA directly into the testis parenchyma of adults (in vivo), or introducing the transformed donor spermatogoniainto testes of a sterile recipient (ex vivo) for further obtaining progeny. In case of introduction of donor spermatogenic cells the key points that determine the effectiveness of the manipulation is to obtain a pure population of donor cells and the elimination of their own spermatogenic cells (spermatogenesis off). In this regard, conducting research related with the development of effective methods for isolating and maintaining the culture of testes stem cells (spermatogonia) is actual. The aim of our study was the optimization of the methodological approaches to the preparation and cultivation of rooster spermatogoniawithin the development of individual stages for producing transgenic birds. In the experiments we obtained and characterized the culture of rooster spermatogonia. Based on preliminary histological studies of spermatogenesis it was found that in roosters aged under 5 weeks the spermatogenic cells are predominantly presented by one cell type - spermatogonia. In this connection, for obtaining spermatogoniaculture the testes from two week old cockerels have been used. Isolation of spermatogenic cells from testicular of rooster was achieved by mechanical and enzymatic treatments of the testis tissue. For enzymatic dissociation of testis tissue we used sequential processing with collagenase solution (at a final concentration of 1 mg/ml) for 20 minutes and 0.25% trypsin solution for 30 minutes. For obtaining the most pure population of spermatogoniawe took into account different capacity of different cell types to adhesion. It was found that after 24 hours of culturing primary culture of rooster testicles the unattached cells were presented mainly by spermatogonia. These cells were pelleted and plated into culture dishes with feeder layers. Continuous cell line STO, transplanted Sertoli cells of swine, a cell line Sc, primary Sertoli cells of rooster were used as feeder layers. Also we carried out the cultivation of spermatogonia on the dishes coated with 0.2% gelatin. The growth medium for culturing spermatogonialcells was DMEM with high glucose content (4.5 g/l), supplemented with 5% FCS (Fetal Calf Serum, GE Healthcare Life Sciences HyCloneLaboratories, USA), 2 mMa-glutamine (Invitrogen, USA), MEM (Minimum Essential Medium, 10 μl/ml, Invitrogen, USA), antibiotics (100х, Invitrogen, USA), mercaptoethanol (5х10-5М, Invitrogen, USA), albumin (5 mg/ml, Invitrogen, USA), DL-lactic acid (1 μl/ml, Sigma-Aldrich Сo., USA), epidermal growthfactorEGF(20 ng/ml, Sigma-Aldrich Сo., USA), basic fibroblast growth factorbFGF (10 ng/ml, Sigma-Aldrich Сo., USA), leukemia inhibitory factorLIF (2ng/ml,Sigma-Aldrich Сo., USA).Spermatogonia colonies have been formed at days 3-4 of cultivation. Best results were obtained by culturing the spermatogonia on primary Sertoli cells of rooster. The presence of spermatogonia colonies was confirmed immunohistochemically in 7-dayculture using SSEA-1 (stage-specific embryonic antigen-1)specific antibodies.