Genetic Variations in the Microsatellite Contribute to the Regulation of Bovine Sperm-Borne Antioxidant Capacity.

Nuclear factor (erythroid-derived 2)-like 2 () is a transcription factor protein-coding gene, considered a master regulator of the cellular stress response. The genetic variations of the could influence its transcriptional profile and, subsequently, the stress resilience in all cell types, including sperm cells. Therefore, the sperm-borne antioxidants abundance in association with the genetic variation of a GCC microsatellite located at the 5' upstream region of the gene was investigated in young ( = 8) and old ( = 8) Holstein bulls' sperm cells at different seasons. The sperm DNA was sequenced using Sanger sequencing, while- the sperm-borne mRNA analysis was carried out using the synthesized cDNA and qPCR. The data were statistically analyzed using GraphPad Prism 10.0.2 software. The results showed that two bulls had a heterozygous genotype of eight and nine GCC repeats, while biallelic of eight, nine, and fifteen repeats were identified in two, ten, and two bulls, respectively. The computational in silico analysis revealed that the upstream sequence with 15, 9, and 8 GCC repeats bound with 725, 709, and 707 DNA-binding transcription factor proteins, respectively. Lower quality of sperm DNA was detected in the spring season compared to other seasons and in young bulls compared to old ones, particularly in the summer and autumn seasons. The mRNA expression analysis revealed that the gene was the abundant transcript among the studied sperm-borne antioxidants and was significantly determined in old bulls' spermatozoa. Moreover, two transcripts of the gene and antioxidant (, , , , ) genes displayed differential expression patterns between the age groups across seasons in an antioxidant-dependent manner. The bulls with a heterozygous GCC sequence exhibited elevated sperm-borne mRNA levels of and transcripts. Taken together, the findings suggest that the -GCC microsatellite may contribute to the transcription regulation of transcripts and their subsequent downstream antioxidants in bovine sperm cells.