2.5. Global Methylation Analysis

DNA for global methylation analysis was intended for analysis taking into account the division into sex of individuals after prior PCR identification and prepared using a commercial kit for methylated DNA quantification (MDQ1, Imprint Methylated DNA Quantification Kit, Sigma-Aldrich) according to the manufacturer’s protocol. DNA isolated from gPGCs (n = 5 for each time point and each sex) was diluted in a binding solution to a final concentration of 150 ng/µL. Then, it was used for estimating the level of methylated DNA based on enzyme-linked immunosorbent assay on 96-well plates. Positive (methylated) and blank controls were analyzed together with the DNA samples. The absorbance was measured at 450 nm by using the Multiscan (Thermo-Fisher Scientific, Waltham, MA, USA) microplate reader and read by SkanIt software 6.0.2. (Thermo-Fisher Scientific, Waltham, MA, USA). For each time point (4.5, 8, and 12 days), five samples per group (male/female), each derived from a different individual, were analyzed. The absorbance measurements were taken in two technical repetitions, each time using the same amount of DNA. The two measurements were averaged, and the mean value was used for the further analysis. Global DNA methylation levels were expressed as percentages relative to the methylated control and were calculated using the following formula: A450S−A450BA450MC−A450B×100%, where A450S is the average absorbance of the sample, A450B is the average absorbance of the blank, and A450MC is the average absorbance of the methylated control. Statistical analysis was carried out by SAS Enterprise Guide 8.2 (SAS Institute Inc., Cary, NC, USA). The quantitative values were first analyzed for normality using the Shapiro–Wilk test. Obtained values failed normal distribution assumption; therefore, the Kruskal–Wallis test was used (the influence of sex and the influence of developmental day; p < 0.05).