302 VIABILITY OF BOVINE SPERMATOZOA INCUBATED WITH FOREIGN DNA

Abstract

Several studies have been performed over the years to promote the understanding and improvement of the sperm-mediated gene transfer technique. However, little is known about the effect of exogenous DNA in the sperm cells. The aim of this study was to evaluate the effect of the incubation period and exogenous DNA addition on mitochondrial activity and acrosomal status of bovine spermatozoa. Frozen-thawed semen was separated by Percoll gradient (45/90%) at 600g for 30 min, and the pellet was resuspended and washed by centrifugation in sperm-TALP at 200g for 5 min. The spermatozoa were resuspended in fertilization medium (without heparin) at a concentration of 5 × 106 spermatozoa/mL and incubated at 39°C, 5% CO2 in air with 500 ng pEYFP-Nuc/mL (Clontech, Mountain View, CA, USA) (DNA group) for 1 and 2 h or without DNA (control group) for 0, 1 and 2 h. JC-1 (Molecular Probes; Invitrogen Brasil, Ltd., Sao Paulo, Brazil) was used to determine mitochondrial potential and fluorsecein isothiogyanate (FITC-PSA; Sigma-Aldrich, Brazil) was used to assess acrosomal status. Two microliters of JC-1 (76.5 μM in DMSO) and 50 μL of FITC-PSA (100 μg/mL in DPBS) were added to 150 μL of IVF medium + 5 × 106 spermatozoa/mL; the mixture was incubated at 25°C for 10 min. DNA incorporation was evaluated by p-EYFP-Nuc PCR amplification. All treatments were repeated ten times and data were analyzed by ANOVA and Tukey test (P < 0.05). The results are described in Table 1. The sperm were classified as: Class 1 (intact acrosome and high mitochondria potential), Class 2 (intact acrosome and low mitochondria potential), Class 3 (reacted acrosome and high mitochondria potential) and Class 4 (reacted acrosome and low mitochondria potential). In both groups, the Class 1 sperm decreased over time, whereas an increase in number was verified for Class 2 sperm. There was no significant difference in numbers among the incubation periods in both groups for Class 3 sperm. However, there was an increase in Class 4 sperm number over time, indicating that the main effect of the incubation period was the loss of mitochondria potential. There was no effect of the exogenous DNA addition on sperm viability in relation to the control group, indicating that the exogenous DNA had no effect on mitochondrial activity and acrosomal status of bovine semen. Table 1. Effect of incubation period and exogenous DNA addition on sperm viability This work was supported by FAPESP 03/10234–7; 03/07456–8.