Effect of pretreatment of ovine sperm on male pronuclear formation and normal fertilization following intracytoplasmic sperm injection

Introduction: During the final stages of mammalian sperm maturation, their nuclear structure becomes progressively condensed and stabilized by the formation of disulfide bonds. Therefore one of the problems in sheep ICSI is the failure or rarely decondensation of sperm chromatins, resulting in a low rate of male pronuclear formation and normal fertilization in artificially activated ovine oocytes matured in vitro. In view of the dependence of sperm nuclear decondensation on the reduction of disulfide bonds, the aim of the present study is to determine how far the efficiency of ovine ICSI (judged by the rate of male and female PN formation and subsequent embryo development) can be improved by sperm pretreatment with DTT, SDS and two-time frozen/thawed. In addition, considering the role of detergents in solubilizing proteins and disruption of sperm membrane, the probable synergistic effect between DTT and SDS on MPN formation is evaluated. Materials and Methods: Sperm were incubated in HSOF-BSA medium containing: I) 5 mM DTT for 20 min, II) 5 mM DTT plus 0.1% SDS for 20 min, and III) 0.1% SDS for 20 min. The sperm were then washed twice in BSA-HSOF and prepared for injection in the same medium. In group IV) after swim up, the sperm without cryoprotectant was transferred to the-20 freezer for 5 min and then plunged directly into liquid nitrogen for 1 min, and thawed immediately in 37 C. This procedure (freezing in liquid nitrogen and thawing at 37 C) was carried out two times. The non-treated sperm served as control. In pretreated and control groups the sperm were prepared in HSOF-BSA for injection. Results: The rate of swollen sperm head in SDS group (N= 63) was higher (p< 0.05) than DTT and SDS+ DTT groups (44.4% vs. 13% and 13.2%, respectively). The proportion of intact sperm head in DTT group (N= 96) was higher (p< 0.05) than other SDS and SDS+ DTT groups (44% vs. 9.5 and 17%, respectively). Sperm head decondecation (male pronucleus formation) in SDS+ DTT (N= 53, 58.5%) was higher than SDS (27%) and DTT (38.5%) groups (p< 0.05) except for the oocytes injected with frozen-thawed (N= 64, 45.3%) sperm. Conclusion: In the current study pretreatment of sperm with DTT+ SDS led to the highest rate of MPN formation among ICSI oocytes. It seems DTT (an agent that specifically reduces disulfide bonds [S= S]) and SDS (an anionic detergent which disrupts non-covalent bonds) could exert their synergistic effect on MPN formation through sperm nuclear decondensation and sperm membrane damage, respectively.