*2.4. Cell Viability*

Cell viability was assessed using a Trypan blue exclusion assay, by counting live and trypan blue-positive gonocytes as previously described [19], and viability was expressed as the mean ± SEM of the percentage of live cells against the total number of gonocytes in 3 independent experiments, each performed with triplicates.

### *2.5. RNA Extraction and Real-Time Quantitative PCR (Q-PCR) Analysis*

Total RNA was extracted from cell pellets using the PicoPure RNA isolaton kit (Arcturus, Mountain View, CA, USA) and digested with DNase I (Qiagen, Santa Clarita, CA, USA), followed by cDNA synthesis with a single-strand cDNA transcriptor synthesis kit (Roche Diagnostics, Indianapolis, IN, USA), as previously described [18,19]. Quantitative real-time PCR (qPCR) was performed using SYBRgreen PCR Master Mix kit (Bio-Rad, Hercules, CA, USA) on a LightCycler 480 (LC480, Roche Diagnostics) [18]. The forward and reverse primers used are provided in Table 1. The comparative Ct method was used to calculate the relative expression of the di fferentiation marker Stra8, and PRDX 1 to 6, using 18S rRNA as housekeeping gene for data normalization. Changes in Stra8 gene expression are expressed as percent of the control values; and given as means ± SEM from 3 or 4 experiments, each using triplicates. PRDX data are expressed in relative gene expression and shown as the means ± SEM from 3 experiments.
