137 Recovery of quail spermatogenesis by donor spermatogonia transplantation

Abstract

Spermatogonia, the stem cell precursors of male germ cells, are used as convenient biological material for the preservation of genetic resources (cryobanks) and the introduction of recombinant DNA (transgenesis). Donor spermatogonia subsequently differentiate into mature germ cells (spermatozoa), which are used to produce offspring. Our laboratory is investigating methods to improve the efficiency of spermatogonial germ cell transplantation in quail. The objectives of this study were to (1) determine the optimal age for spermatogonia isolation from the testes of donor male quail and (2) identify the most appropriate concentration of busulfan for treatment of recipient quail testes. Statistical analysis was performed using SPSS ver. 15.0 (IBM Corp.; analysis of variance test). In order to determine the optimal age for spermatogonia isolation, testes from male quail at 1-4 weeks of age were isolated and histological studies were performed on a population of spermatogenic cells in the seminiferous tubules. Histological studies of quail testes isolated at different ages showed that the optimal age for obtaining a culture of spermatogonia is a period from 1-2 weeks of age. During this period, spermatogenic cells were represented mainly by spermatogonia (P < 0.01). Therefore, testes of 1-week-old quails were used to obtain a culture of spermatogonia. The resulting cell culture consisted mainly of spermatogonia (85%) with a small number of Sertoli cells. Next, a series of experiments introducing busulfan into quail testes was carried out using concentrations from 10-150 mg kg⁻¹ of liveweight. Experiments showed that an effective dose to remove the recipient male's own spermatogenic cells was a concentration of 100 mg kg⁻¹ of liveweight (P < 0.05). Finally, using the optimal parameters described above, spermatogonia cultures were obtained and introduced into the testes of quail recipients (n = 6), following administration of busulfan for 2-3 weeks before donor spermatogonia were introduced. The effectiveness of spermatogenesis recovery was assessed based on the analysis of sperm from quail recipients at 3 months after the injection of donor cells. The presence of donor germ cells in the testes of quail recipient drakes was confirmed by microsatellite analysis of DNA isolated from the blood and sperm of recipients as well as the donor cells (spermatogonia). The microsatellite profiles of the blood and sperm DNA in quail recipient males were different, which confirms the restoration of spermatogenesis in the studied recipients due to the development of donor germ cells.

The reported study was funded by RFBR, project number 18-29-07079.