The effect of poly L-lactic acid nanofiber on the induction of colony formation of frozen-thawed bovine spermatogonial stem cells in vitro

Preparing a suitable condition for in vitro culture of spermatogonial stem cells can help discover all of the molecular mechanisms involving in spermatogenesis and pave the way for gene manipulation. This study aimed to evaluate the effect of a poly L-lactic acid (PLLA) nanofiber on the proliferation of frozen-thawed bovine spermatogonial stem cells in vitro. The isolated spermatogonial cells from the prepubertal bull were frozen-thawed and cultured in two groups: the control group (C) including the spermatogonial cells and the treatment group (T) including the spermatogonial cells seeded onto PLLA. The cells in both groups were cultured in DMEM supplemented with 5% fetal bovine serum and 40 ng/ml glial cell line-derived neurotropic factor (GDNF) for 2 weeks. Colony assay was conducted on days 4, 7, 10 and 13 after the beginning of the culture. Expression of spermatogonial genes (PLZF, BCL6, GFRα-1, VASA, Itgα6) in the culture was determined by reverse transcription polymerase chain reaction (RT-PCR) on the last day of the experiment. The viability rates of fresh cells before and after the addition of cryoprotectant agent were 87.4±2.4 and 81.8±3.1, respectively which decreased to 65±1.7 after the thawing (P<0.01). Moreover, the results showed that the surface area of colonies derived from spermatogonia were significantly greater in the treatment group compared to the control group on day 13 (P<0.05). Finally, RT-PCR revealed the expression of all spermatogonial stem cell (SSC) markers in both groups. The PLLA nanofiber can provide a suitable microenvironment for frozen-thawed SSCs in an in vitro-culture system.