Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2016, V. 51, № 4, pp. 450-458.

doi: 10.15389/agrobiology.2016.4.450eng

UDC 636.52/.58:576.3/.7.086.83:591.04

Acknowledgements:
Supported financially by Russian Scientific Foundation (grant № 16-16-10059).

 

ISOLATION AND CHARACTERIZATION OF ROOSTER (Gallus gallus)
SPERMATOGONIA

N.A. Volkova, S.V. Korzhikova, T.O. Kotova, A.N. Vetokh, L.A. Volkova,
N.A. Zinovieva

L.K. Ernst All-Russian Research Institute of Animal Husbandry, Federal Agency of Scientific Organizations, pos. Dubrovitsy, Podolsk Region, Moscow Province, 142132 Russia,
e-mail natavolkova@inbox.ru

Received May 30, 2016

 

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 spermatogonia into 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 spermatogonia within 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 spermatogonia culture 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 spermatogonia we 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 spermatogonial cells was DMEM with high glucose content (4.5 g/l), supplemented with 5 % FCS (Fetal Calf Serum, GE Healthcare Life Sciences HyClone Laboratories, USA), 2 mM α-glutamine (Invitrogen, USA), MEM (Minimum Essential  Medium, 10 μl/ml, Invitrogen, USA), antibiotics (100×, Invitrogen, USA), mercaptoethanol (5×10-5 М, Invitrogen, USA), albumin (5 mg/ml, Invitrogen, USA), DL-lactic acid (1 μl/ml, Sigma-Aldrich Сo., USA), epidermal growth factor EGF (20 ng/ml, Sigma-Aldrich Сo., USA), basic fibroblast growth factor bFGF (10 ng/ml, Sigma-Aldrich Сo., USA), leukemia inhibitory factor LIF (2 ng/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-day culture using SSEA-1 (stage-specific embryonic antigen-1) specific antibodies.

Keywords: spermatogonia, spermatogenic cells, roosters, cell culture.

 

Full article (Rus)

Full text (Eng)

 

REFERENCES

  1. Yu F., Ding L.J., Sun G.B., Sun P.X., He X.H., Ni L.G., Li B.C. Transgenic sperm produced by electrotransfection and allogeneic transplantation of chicken fetal spermatogonial stem cells. Mol. Reprod. Dev., 2010, 77: 340-347 CrossRef
  2. Brinster R.L. Germline stem cell transplantation and transgenesis. Science, 2002, 296: 2174-2176 CrossRef
  3. 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.
  4. Zheng Y., Zhang Y., Qu R., He Y., Tian X., Zeng W. Spermatogonial stem cells from domestic animals: Progress and prospects. Reproduction, 2014, 147: 65-74 CrossRef
  5. McLean D.J. Spermatogonial stem cell transplantation and testicular function. Cell Tissue Res., 2005, 322(1): 21-31 CrossRef
  6. Gistologiya: atlas, uchebnoe posobie /Pod redaktsiei V.L. Bykova, L.K. Zhunkeira, Zh. Karneiro [Hystology: Atlas. V.L. Bykov, L.K. Zhunkeir (eds.)]. Moscow, 2009 (in Russ.).
  7. De Rooij D.E., Griswold M.D. Questions about spermatogonia posed and answered since 2000. J. Androl., 2012, 33: 1085-1095 CrossRef
  8. De Rooij D.G., Russell L.D. All you wanted to know about spermatogonia but were afraid to ask. J. Androl., 2000, 21: 776-798 CrossRef
  9. Volkova N.A., Volkova L.A., Fomin I.K., Zinovieva N.A., Lotsmanova N.S. Optimization of conditions for recombinant DNA injection into spermatogenic cells of the chicken in vivo. Agricultural Biology, 2012, 6: 56-60 (in Engl.) CrossRef
  10. Min S., Qing S.Q., Hui Y.Y., Zhi F.D., Rong Q.Y., Feng X., Chun L.B. Generation of antiviral transgenic chicken using spermatogonial stem cell transfected in vivo. African Journal of Biotechnology, 2011, 10(70): 15678-15683 CrossRef
  11. Dobrinski I. Germ cell transplantation and testis tissue xenografting in domestic animals. Anim. Reprod. Sci., 2005, 89: 137-145 CrossRef
  12. Rodriguez-Sosa J.R., Dobson H., Hahnel A. Isolation and transplantation of spermatogonia in sheep. Theriogenology, 2006, 66: 2091-2103 CrossRef
  13. 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: 612-616 CrossRef
  14. Nagano M., Ryu B.Y., Brinster C.J., Avarbock M.R., Brinster R.L. Maintenance of mouse male germ line stem cells in vitro. Biol. Reprod., 2003, 68: 2207-2214 CrossRef
  15. 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: 611-617 CrossRef
  16. 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: 26333-26346 CrossRef
  17. 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: 983-991 CrossRef
  18. Savchenkova I.P., Vasil'eva S.A. Tsitologiya, 2016, 58(2): 135-142 (in Russ.).
  19. Goel S., Sugimoto M., Minami N., Yamada M., Kume S., Imai H. Identification, isolation, and in vitro culture of porcine gonocytes. Biol. Reprod., 2007, 77: 127-137 CrossRef
  20. Han S.Y., Gupta M.K., Uhm S.J., Lee H.T. Isolation and in vitro culture of pig spermatogonial stem cell. Asian-Aust. J. Anim. Sci., 2009, 22: 187-193 CrossRef
  21. Pramod R.K., Mitra A. In vitro culture and characterization of spermatogonial stem cells on sertoli cell feeder layer in goat (Sapra hircus). J. Assist. Reprod. Genet., 2014, 31: 993-1001 CrossRef
  22. Heidari B., Rahmati-Ahmadabadi M., Akhondi M.M., Zarnani A.H., Jeddi-Tehrani M., Shirazi A., Naderi M.M., Behzadi B. Isolation, identification, and culture of goat spermatogonial stem cells using c-kit and PGP9.5 markers. J. Assist. Reprod. Genet., 2012, 29: 1029-1038 CrossRef
  23. 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 CrossRef
  24. 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 CrossRef
  25. 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 CrossRef
  26. Beloglazova E.V., Kotova T.O., Volkova N.A., Volkova L.A., Zinovieva N.A., Ernst L.K. Age dynamics of spermatogenesis in cocks in connection with optimization of bioengineering manipulation time. Agricultural Biology, 2011, 6: 60-64 (in Engl., in Russ.). Available http://www.agrobiology.ru/6-2011beloglazova-eng.html, http://www.agrobiology.ru/6-2011beloglazova.html. No date.
  27. Mikroskopicheskaya tekhnika /Pod redaktsiei D.S. Sarkizova, Yu.P. Perova [Microscopy. D.S. Sarkizov, Yu.P. Petrov (eds.)]. Moscow, 1996.

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