221 Isolation of turkey spermatogonia

Abstract

Across different types of spermatogenic epithelium, researchers have focused on the study and production of spermatogonial stem cells and their use as genetic material for creating gene pool cryobanks of valuable breeds and lines of poultry. The aim of our research was to identify optimal conditions for obtaining and culturing turkey spermatogonia. A series of experiments were carried out to determine the optimal age of males for isolating spermatogonia and optimize conditions for their isolation and cultivation. Age-related features of turkey spermatogenesis were assessed by testis histological studies. Statistical analysis was performed using SPSS v15.0 (t-test; SPSS Inc./IBM Corp.). Turkey testis tissue was mechanically crushed and treated with enzymes: 0.1% collagenase and 0.25% trypsin. Purification of spermatogonia from other types of cells was performed by adhesion and separation. The conditions and duration of spermatogenic cell cultures were selected experimentally. Primary Sertoli cells of turkeys, Sertoli cells of roosters, STO cell line, and transplanted Sertoli cells of pigs (PTP) were used as a feeder layer for the cultivation of spermatogonia. Spermatogonia culture medium consisted of Dulbecco's modified Eagle's medium (DMEM)-high glucose supplemented with 5% fetal calf serum, 2 mM α-glutamine, 10 µL mL⁻¹ MEM, 100x antibiotics, 10 µL mL⁻¹ ITS solution, 5 × 10⁻⁵ M mercaptoethanol, 5 mg mL⁻¹ albumin, 1 µL mL⁻¹ DL-lactic acid, 20 ng mL⁻¹ epidermal growth factor, 10 ng mL⁻¹ basic fibroblast growth factor, and 2 ng mL⁻¹ leukemia inhibitory growth factor (LIF). The SSEA-1 antibody was used for identification of spermatogonia colonies. Histological study of the turkey testes at different ages showed that in animals under 12 weeks, the population of generative cells was represented mainly by spermatogonia (P < 0.05). Thus, spermatogonia were isolated from the testes of 4-wk-old males. Isolated cells were cultured for 24 h, after which the supernatant containing non-adherent cells (mainly spermatogonia) was transferred to a new dish for continued culture. Maximal homogeneity of the cell population was detected after transferring non-adherent cells 3 times at 24-h intervals. The best cell growth was observed when spermatogonia were cultivated on turkey Sertoli cells. The number of these cells in suspension reached 81% purity. Formation of spermatogonia colonies was observed on Days 7 to 8 of cultivation, the presence of which was confirmed by immunohistochemical staining with SSEA-1.

This study was supported by the Russian Science Foundation within project no.16-16-04104.