201 Spermatogonia Transplantation in the Chicken

Spermatogonia are the precursors of male germ cells. They are a valuable genetic material for the production of transgenic poultry. This technology includes isolation of the spermatogonia from male donor’s testes, transformation, and transplantation of donor cells into the sterilized recipient’s testes. The transplanted spermatogonia subsequently differentiate into male sex cells (sperm). The aim of this study was to optimize the individual stages of donor spermatogonia transplantation into the recipient’s testes to increase the effectiveness of spermatogenesis recovery. In the first stage, the spermatogenesis in male chicken was examined to determine the optimal age for isolation of spermatogonia from testes. Histological examinations of male chicken testes (n = 80 birds) were done for 8 age categories, from 1 week to 3 months. It was found that under the age of 4 weeks, the cell population in the seminiferous tubules of male chickens was represented mainly by Sertoli cells and spermatogonia. Maximum percentage of spermatogonia was 69 ± 3% at 4 weeks. At the next stage, a culture of spermatogonia was obtained. Testes of 3-week-old male chickens were used. Separation of the spermatogonia from other types of cells was based on a differential adhesive capacity. The maximum homogeneity of the cell population was established by transfer (3 times) of the supernatant containing unattached cells after 24 h of cultivation into a new culture dish for further cultivation. The cell population is represented mainly by the spermatogonia (89 ± 3%). The lentiviral transduction (pHAGE vector, ZsGreen under CMV promotor) was used to transform the resulting culture of the spermatogonia. The efficiency of spermatogonia infection with lentiviral particles (TU/mL = 2.5 × 108) was 65 ± 2%. After transformation, spermatogonia were introduced into the testes of busulfan-sterilized recipients. The optimal concentration of busulfan treatment after series of experiments from 40 to 100 mg/kg was determined. The effective dose for the removal of own spermatogenic cells was revealed at a concentration of 80 mg/kg of live weight. With complete elimination of other types of spermatogenic cells, the number of Sertoli cells and spermatogonia in the testicle tubules decreased by 39 ± 2% and 98 ± 1%, respectively, compared with the control group. The efficiency of spermatogenesis recovery was assessed based on sperm analysis that was obtained from male recipients (n = 5 birds) 4 months after the introduction of donor cells using PCR. The presence of recombinant DNA (ZsGreen) in recipients’ sperm was shown. Thus, our results indicate the prospect of using spermatogonia as a genetic material for the production of transgenic poultry. Study was supported by the Russian Science Foundation (Project no.16-16-10059).