Basic fibroblast growth factor (bfgf)

bFGF (2 μg; ABclonal Biotechnology Co., Ltd.) was dissolved in 333 μL of 5 mM Tris (pH 7.6) containing 0.1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO), mixed with 7 mL of DAFM/chitosan solution and then formed into hydrogels as described above. To study bFGF release kinetics, DAFM/chitosan hybrid hydrogels containing bFGF were placed in 30 mL of PBS and incubated at 37°C, and samples of the medium were collected at various time points. The concentration of free bFGF in the medium was quantified by enzyme-linked immunosorbent assay (ELISA) kit (ABclonal Biotechnology Co., Ltd.) according to the manufacturer's instructions. Absorbance was read on a microplate reader (BioTek Instruments) at 450 nm.

The culture and transfection of spermatogonia from the fat greenling were conducted following standardized protocols. The spermatogonia were cultured in L-15 medium supplemented with 10 ng/mL bovine insulin (Solarbio, Beijing, China), 1nM sodium pyruvate (Solarbio, Beijing, China), 1% fish serum, 10 ng/mL glial cell line-derived neurotrophic factor (GDNF, ABclonal, Wuhan, China), 10 ng/mL basic fibroblast growth factor (bFGF, ABclonal, Wuhan, China), and 5% fetal bovine serum (FBS).
For the transfection procedure, cells at the optimal confluence were transfected with prf1-pEGFP-N1 and gzmb-pEGFP-N1 plasmids in the experimental group, and with the empty pEGFP-N1 plasmid in the control group, using Lipofectamine 3000 Transfection Reagent (Catalog No. L3000015, Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. Post-transfection, cells were maintained under standard culture conditions and monitored for expression of the transgene. Total RNA was extracted from spermatogonia of both the experimental and control groups, and cDNA was synthesized. The expression of prf1, gzmb, and apoptosis-related genes was analyzed by qRT-PCR (following the procedure described in Section 4.4). The primer sequences are listed in Table S1.