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Sperm nuclear proteins, the protamines (PRM) and transition nuclear proteins (TNP) play a crucial role in sperm nuclear condensation. The compact packaging of sperm DNA by protamines maintains sperm genome integrity, which is prerequisite for normal sperm function. However the effect of nucleotide variations in PRM and TNP genes on sperm DNA integrity and male fertility is not clear. This case-control study was planned to analyze PRM and TNP gene nucleotide variations and sperm DNA integrity in 100 oligozoospermic infertile men and 100 fertile controls. Protamine and TNP genes were amplified by polymerase chain reaction and sequenced. Flow cytometry-sperm chromatin structure assay (FC-SCSA) was applied to measure the DNA fragmentation index (DFI) in sperm. Semen analysis was performed as per WHO [1999] guidelines with slight modification. In total, 7 nucleotide variations including two novel changes, a non-synonymous mutation in the exon-2 of PRM2 gene (c.443C > A) and a novel insertion of T (c.396_397InsT) at the 3' UTR region of TNP1 were detected. None of the nucleotide changes were observed with increased risk frequency in the oligozoospermic infertile men compared to the controls. Though overall DFI was significantly (p < 0.0001) higher in infertile men compared to controls (36.31 ± 7.25 vs. 26.49 ± 2.78) irrespective of nucleotide changes, no such difference was observed between 100 infertile men or pooled population of 200 with and without mutations. However it was observed that two cases with novel nucleotide changes PRM2 c.443C > A and TNP1 c.396_397InsT had higher DFI value of 34.82% and 43.85%, respectively. In conclusion, our pilot study for the first time in the Indian population revealed two rare novel mutations in sperm nuclear protein genes that are perhaps associated with higher sperm DNA fragmentation.

Purpose To determine the effects of α-tocopherol supplementation to oocyte maturation media and embryo culture media on the yield of ovine embryos. Methods α-tocopherol, at concentrations of 0, 50, 100, 200, 400 and 500 µM was supplemented to ovine oocyte or embryo culture media and cultured at 5% or 20% O 2 levels. Percentages of cleavage, morula and blastocyst, total cell count and comet assay were taken as indicators of developmental competence of embryos. Results 200 µM α-tocopherol in embryo culture medium at 20% O 2 level significantly increased the rates of cleavage (P<0.05), morulae (P<0.05) and blastocyst (P<0.01) formation and blastocyst total cell number (P<0.01) when compared with control. The rates of blastocyst formation were also significantly higher in 100 µM (P<0.01) and 400 µM (P<0.05) supplemented groups than control. Conclusion α-tocopherol supplementation may enhance the in vitro developmental competence of ovine embryos by protecting them from oxidative damage.

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