Obtaining and characterization of the spermatogenic cell culture of males from interspecific hybrids of domestic sheep and argal

   Relevance. The creation of biological material cryobanks is one of the key methods for the conservation and maintenance of the biodiversity of animal genetic resources. The biomaterial widely used for preservation in cryobank conditions are mature germ cells of males – spermatozoa. As a promising alternative for these purposes is considered the use of testis stem cells – spermatogonia which makes it possible to select biomaterial from immature animals with a valuable genotype. The article presents data on obtaining a culture of spermatogonia of males of interspecific hybrids of domestic sheep with argali.
   Methods. The object of research was spermatogenic cells of sheep's interspecific hybrids from the Romanov breed with argali. The testes of hybrid males served as a material for obtaining a spermatogenic cells culture. The conditions for isolating and maintaining spermatogonia in culture in vitro were optimized using histological, cytological, immunohistochemical and cultural methods.
   Results. It has been established that the effectiveness of obtaining a spermatogenic cells culture, maximally enriched with spermatogonia, are affected by the age of the males from which the biomaterial is taken, the preliminary purification of spermatogonia from other types of spermatogenic and somatic testicular cells, the growth medium and the type of feeder layer used for the cultivation of spermatogonia. It is shown that the optimal age of males for the selection of biomaterial is the age period from birth to 4 months. During this period, the cells of the epitheliospermatogenic layer in the seminiferous tubules of the testes from hybrid males are mainly represented by one type of spermatogenic cells – spermatogonia (92–100 %). The maximum purification of spermatogonia from other types of cells is achieved by separating them according to adhesion. High intensity of growth and formation of spermatogonia colonies is observed when they are cultivated on the feeder layer formed by the primary culture of own Sertoli cells, as well as Sertoli cells from another rams. Under these conditions, the attachment of spermatogonia to the cells of the feeder layer is noted on the 1st – 2nd day of cultivation, the formation of colonies – on the 6th day of cultivation.

1. Boettcher P. J., Akin O. Current arrangements for national and regional conservation of animal genetic resources. Animal Genetic Resources. 2010; (47): 73–83. doi: 10.1017/S2078633610000949

2. Singina G. N., Volkova N. A., Bagirov V. A., Zinovieva N. A. Cryobanking of somatic cells in conservation of animal genetic resources: prospects and successes (review). Agricultural Biology. 2014; 6: 3–14. doi: 10.15389/agrobiology.2014.6.3eng

3. Silversides F. G., Purdy P. H., Blackburn H. D. Comparative costs of programmes to conserve chicken genetic variation based on maintaining living populations or storing cryopreserved material. Br Poult Sci. 2012. 53 (5). P. 599-607 375. doi: 10.1080/00071668.2012.727383

4. Mara L., Casu S., Carta A., Dattena M. Cryobanking of farm animal gametes and embryos as a means of conserving livestock genetics. Anim Reprod Sci. 2013; 138 (1–2): 25-38. doi: 10.1016/j.anireprosci.2013.02.006

5. Zheng Y., Zhang Y., Qu R., He Y., Tian X., Zeng W. Spermatogonial stem cells from domestic animals: Progress and prospects. Reproduction. 2014; 147 (3): 65–74. doi: 10.1530/REP-13-0466

6. Kanatsu-Shinohara M., Ogonuki N., Inoue K., Miki H., Ogura A., Toyokuni S., Shinohara T. Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol. Reprod. 2003; 69 (2): 612–616. doi: 10.1095/biolreprod.103.017012

7. Kanatsu-Shinohara M., Muneto T., Lee J., Takenaka M., Chuma S., Nakatsuji N., Horiuchi T., Shinohara T. Long-term culture of male germline stem cells from hamster testes. Biol. Reprod. 2008; 78 (4): 611–617. doi: 10.1095/biolreprod.107.065615

8. Wang X., Chen T., Zhang Ya., Li B., Xu Q., Song C. Isolation and culture of pig spermatogonial stem cells and their in vitro differentiation into neuron-like cells and adipocytes. Int. J. Mol. Sci. 2015; 16 (11): 26333-26346. doi: 10.3390/ijms161125958

9. Park M. H., Park J. E., Kim M. S., Lee K. Y., Park H. J., Yun J. I., Choi J. H., Lee E., Lee S. T. Development of a high-yield technique to isolate spermatogonial stem cells from porcine testes. J. Assist. Reprod. Genet. 2014; 31 (8): 983–991. doi: 10.1007/s10815-014-0271-7

10. Oatley J. M. Spermatogonial stem cell biology in the bull: development of isolation, culture, and transplantation methodologies and their potential impacts on cattle production. Soc. Reprod. Fertil. Suppl. 2010; 67: 133–143.

11. Pramod R. K., Mitra A. In vitro culture and characterization of spermatogonial stem cells on sertoli cell feeder layer in goat (Сapra hircus). J. Assist. Reprod. Genet. 2014; 31 (8): 993–1001. doi: 10.1007/s10815-014-0277-1

12. Sisakhtnezhad S., Bahrami A. R., Matin M. M., Dehghani H., Momeni-Moghaddam M., Boozarpour S., Farshchian M., Dastpak M. The molecular signature and spermatogenesis potential of newborn chicken spermatogonial stem cells in vitro. In Vitro Cell Dev. Biol. Anim. 2015; 51: 415–425. doi: 10.1007/s11626-014-9843-1

13. Li B., Wang X. Y., Tian Z., Xiao X. J., Xu Q., Wei C. X., Sun H. C., Chen G. H. Directional differentiation of chicken spermatogonial stem cells in vitro. Cytotherapy. 2010; 12 (3): 326–331. doi: 10.3109/14653240903518155

14. Lacerda S. M., Costa G. M., de Franca L. R. Biology and identity of fish spermatogonial stem cell. Gen. Comp. Endocrinol. 2014; 207: 56–65. doi: 10.1016/j.ygcen.2014.06.018