230 EFFECT OF DIFFERENT CONCENTRATIONS OF GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR ON EXPRESSION OF SELF-RENEWAL RELATED GENES IN GOAT (CAPRA HIRCUS) SPERMATOGONIAL STEM CELLS

Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-β superfamily produced by Sertoli cells, is essential for the self-renewal of spermatogonial stem cells in vivo. The present study evaluated the effects of different concentrations of GDNF (human recombinant expressed in Escherichia coli) on expression of some self-renewal related genes in spermatogonial stem cells (SSC). The SSC were isolated from prepubertal goat testes (3–6 months of age) by using double enzymatic digestion method and filtration through 80- and 60-µm nylon mesh filters. Further enrichment was achieved by differential plating on Datura stramonium agglutinin (DSA) lectin-coated dishes and Percoll density gradient centrifugation. The enriched cells were cultured on goat Sertoli cell feeder layer in DMEM + 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Primary SSC colonies were formed within 7 to 10 days. These colonies were characterised through alkaline phosphatase and immunofluorescence staining on Day 10 for different SSC-specific protein markers. Colonies were found to be positive for DBA, THY1, PLZF, UCHL1, OCT-4, SOX2, and NANOG, and negative for c-Kit expression. These colonies were cultured for 15 days without or with supplementation of GDNF forming following groups: (1) without GDNF (control), (2) 10 ng mL–1 GDNF, (3) 20 ng mL–1 GDNF, and (4) 40 ng mL–1 GDNF. RNA was isolated from 100 colonies from 3 different trials on Day 15 of culture, and relative expression of different self-renewal related genes was determined by qRT-PCR. Relative mRNA abundance of PLZF was higher (P < 0.05) following supplementation with 40 ng mL–1 GDNF than in other groups (i.e. control, 10 and 20 ng mL–1 GDNF). Expression of BCL6B and ID4 was found to be significantly higher (P < 0.05) after supplementation of GDNF at all concentrations compared with the control group. Expression of UCHL1 was higher with addition of 20 and 40 ng mL–1 GDNF (P < 0.05), whereas expression of THY1 was higher with supplementation of 10 ng mL–1 GDNF (P < 0.05). In conclusion, GDNF was found to benefit expression of goat SSC candidate genes at a concentration of 40 ng mL–1.