Evaluation of the Effect of L- arginine on Induction of Ovine Spermatogonial Stem Cells Colonization In-Vitro

Spermatogonial stem cells have the ability to self-renewal and differentiation and can transmit genetic traits to the next generation. Therefore, establishing an appropriate culture medium for this cell line is important. The aim of this study was to determine the effect of different concentrations of L-arginine on sheep spermatogonial stem cells colony formation in-vitro.

An experimental study was conducted. Each treatment was replicated five times. Spermatogonial cells were isolated from prepubertal lamb’s testis using two-step enzymatic digestion. The cells were cultured for ten days in six groups. In the control group, a simple culture of spermatogonial cells was performed in DMEM containing 1% antibiotics and 5% FBS. In the treatment groups 1, 2, 3, 4, and 5 different concentrations of L-arginine (50, 100, 200, 500, and 1000 μmol/L), was added to the culture medium, respectively. The culture media were changed every three days. Identification of cells was confirmed by immunocytochemical staining against PGP9.5 and PLZF antigens. Immediately after isolation, the percentage of cells viability, number, and surface area of colonies formed on the 4th, 7th, and 10th days after the culture, were assessed by an inverted microscope. Data were analyzed using one- way ANOVA test. P<0.05 was considered statistically significant.

The findings indicated that the viability rate of spermatogonial cells after isolation was 89.28 ± 1.4%. Isolated cells expressed PGP9.5 and PLZF antigens. Also, on day 10 after culture and in treatment 4 (200 μmol/L L- arginine); the number (160.8 ± 21.1) and surface area (5.4 ± 1.3) spermatogonial cells colonies had a significant increase compared to other groups (P<0.05).

It seems that the addition of L- arginine at a dose of 200 μmol/L has a positive effect on the induction of spermatogonial stem cells colonization and can provide an appropriate culture medium in vitro.