142 Isolation of the Quail Spermatogonia

Spermatogonia are testicular stem cells, the precursors of male sex cells. They are target cells for introduction of recombinant DNA and suitable for creation of cryobanks to preserve biological materials. The aim of our research was to optimize the individual stages culturing quail spermatogonia. In an initial study, dynamics of change in the composition of spermatogenic cells in the seminiferous tubules were assessed histologically, at weekly intervals from 1 week to 1.5 months of age. Thereafter, spermatogonia were isolated from quail testes. Disaggregation of the testis tissue was carried out by consecutive enzymatic treatment in 0.25% trypsin and 0.1% collagenase solution. Purification of spermatogonia from other types of spermatogenic cells was conducted by separation of the cells by adhesion. The duration and conditions of cultivation of spermatogenic cells were selected experimentally. Cultivation of spermatogonia was performed on feeder layers, including quail primary Sertoli cells, STO cell line, and transplanted porcine Sertoli cells. Growth medium for culturing spermatogonia was DMEM HG medium supplemented with 5% FCS, 2 mM α-glutamine, MEM (10 μL mL−1), antibiotic (100×), insulin-transferrin-selenium (ITS, 10 μL mL−1), 2-mercaptoethanol (5 × 10−5 M), albumin (5 mg mL−1), epidermal growth factor (EGF, 20 ng mL−1), basic fibroblast growth factor (bFGF, 10 ng mL−1), and leukemia inhibitory factor (LIF, 2 ng mL−1). For identification of spermatogonia colonies, SSEA-1 antibodies were used. The maximum number of spermatogonia in seminiferous tubules of quail occurred at 3 weeks of age; there were mainly spermatogonia and Sertoli cells at this time. The percentage of spermatogonia from the total number of spermatogenic cells in the seminiferous tubule reached 76 ± 2%. In view of this, spermatogonia were isolated from the testes of 2-week-old quail. Spermatogenic cells were cultured for 24 h, after which the supernatant with unattached cells, mainly spermatogonia, was transferred to a new dish and cultured. Maximum homogeneity of the cell population was detected by dividing the cells by 3-fold transfer of the cell supernatant at an interval of 24 h; the proportion of spermatogonia in the suspension reached 88%. Quail Sertoli cells were the optimal feeder layer for cultivation of quail spermatogonia. Formation of spermatogonia colonies was observed on Day 5 to 7 of cultures, and their identity confirmed by immunohistochemical staining for SSEA-1. The study was supported by the Russian Science Foundation within Project no.16-16-04104.