P-16: Antiapoptotic and Antioxidant Effect of Insulin Like Factor-3 on Sperm of Fertile Men After Cryopreservation
Freezing process has destructive effect on different functions of sperm. Therefore, sperm motility was reduced and apoptosis and DNA damage and also levels of reactive oxygen species (ROS) were increased. Insulin-like factor-3 (INSL3) is a member of Relaxin family and directly affects on spermatogenesis and also plays a protective role against apoptosis. Therefore, with due regard to harmful effects of freezing on sperm and antiapoptotic role of INSL3 in male reproductive system, this study was designed to study the effects of this hormone in reducing complications from freezing.
Materials and methods
Semen samples were collected from 15 fertile men. Each sample was divided into six parts. One part of sample was kept as control group and insulin-like 3 (INSL3) hormone with concentrations of 10, 100, 250, 500 and 1000 ng/ml was added to other five parts. We assayed the level of DNA damage through TUNEL method, activated Caspase enzymes with FLICA marker and reactive oxygen species in mitochondria and cytosol with MSR and DHE markers through flowcytometry method, respectively. And, also motility and morphology of sperm were studied after two weeks freezing.
The level of DNA damage in 250 ng/ml concentration in compare with control group has significant reduction and also activation level of Caspases in 100 ng/ml concentration in compare with 1000 ng/ml concentration has significant increase (P≤0.05). However, significant differences were not observed in different concentrations of hormone concerning motility, morphology and ROS level in mitochondria and cytosol.
This study demonstrated that the effect of INSL3 was completely dose-dependent. But, generally, 250 ng/ml concentration caused decrease in DNA damage level in comparison to the control group and it seems that 250 ng/ml concentration is a suitable concentration for protecting sperm in freezing medium.