Dynamics of Sperm Chromatin Associated with Bull Fertility.

Abstract

Genomic DNA of bull spermatozoa is tightly packaged mostly around Protamine 1 (PRM1) and small amounts of histones such as histone3 (H3) in a much smaller volume compared to somatic cells. Proper packaging of genome is important both for sperm morphology and physiology as well as for fertilization and embryonic development. Both PRM1 and histone3.3 (H3.3; a variant of H3) are known to play important roles during sperm chromatin condensation. Recently, H3.3 has been shown to be bound to actively transcribed regions of the paternal genome; therefore, its presence becomes important. However, molecular and cellular underpinnings of their functions in bull spermatozoa and how these two proteins regulate fertility are not known. The objective of this study was to determine the dynamics of chromatin structure and the expression patterns of DNA binding proteins PRM1 and H3.3 in spermatozoa from bulls with varying fertility. To examine sperm chromatin condensation, chromatin integrity and protamination were detected using halomax assay and toluidine blue staining in spermatozoa from 20 bulls with distinct fertility, which was equally grouped into low and high fertility. For both methods, three biological and two technical replicates were performed per bull, and 500 spermatozoa were counted per slide by two different technicians using light microscopy. We also performed western blotting to detect expression levels of PRM1 and H3.3 followed by acetic acid-urea gel system and computational biology to determine functional motifs in the two proteins. In addition, spermatozoa from three bulls per group were used to conduct IVF experiments with three replicates. Statistical analysis was performed using SAS. Our results revealed that sperm chromatin integrity 7.01% (±2.24) vs. 3.97% (±0.95); p < 0.0001 and protamination 3.27% (±0.99) vs.1.2% (±0.91); p < 0.0001 were significantly different between low and high fertility groups, respectively. Based on the IVF results, embryo cleavage and development rates to blastocyst stage were lower in low fertility group compared to their high fertility counterparts 57.56% (±10.21) vs. 68.92% (±11.65); p <0.05 and 11.7% (± 3.50) vs. 16.96% (± 6.51); p < 0.05, respectively. We confirmed that slide readings of halomax and toluidine blue experiments were not different between the two technicians (p > 0.54 and > 0.80, respectively). We also found that in vivo fertility scores of the bulls were negatively correlated with sperm chromatin protamination (r= −0.62; p < 0.0001) and integrity (r= −0.69; p < 0.0001). In addition, sperm chromatin integrity among the bulls was negatively associated with embryo cleavage (r= −0.56; p < 0.05) and development rate (r= −0.45; p = 0.0637) in embryos derived using spermatozoa from the bulls with varying fertility. In contrast to PRM1, functional motifs were only found in H3 such as histone H3.2, CAMP phospho site, myristyl and Ck2 phospho. We also showed that there is a ratio between the protein expression levels of PRM1 and H3.3 among the bulls. In conclusion, our data showed that inadequate sperm chromatin protamination and integrity were associated with inefficient sperm chromatin condensation leading to improper fertilization and beyond, which can be estimated using bull fertility scores prior to use them for AI. Our results provide a comprehensive output in sperm chromatin dynamics that impacts sperm viability, fertilization and early embryonic development.