398 EFFECT OF INCUBATION PERIOD ON TRANSFECTION RATES IN BOVINE SPERMATOZOA
M. E. O. A. Assumpção A , W. B. Feitosa A , M. Rovegno A , M. G. Marques A , A. C. Nicacio A , R. Simões A and J. A. Visintin AADepartment of Animal Reproduction – FMVZ, Sao Paulo University, Sao Paulo, SP, Brazil
Reproduction, Fertility and Development 19(1) 314-315 https://doi.org/10.1071/RDv19n1Ab398
Submitted: 12 October 2006 Accepted: 12 October 2006 Published: 12 December 2006
Abstract
Current data about the kinetics of exogenous DNA interaction with spermatozoa are controversial. The amount of DNA associated with the spermatozoa is proportional to the incubation period. However, a great amount of exogenous DNA, associated or internalized, can activate endonucleasis, which cleaves the exogenous and/or endogenous DNA similarly to apoptosis. The aim of this work was to evaluate the effect of the incubation period of bovine spermatozoa with exogenous DNA on transfection rate and induction of apoptosis, oncosis, or initial oncosis and/or late apoptosis of sperm cells. For that, 5 × 106 spermatozoa/mL were incubated with 500 ng of plasmid pEYFP-Nuc (Clontech, Mountain View, CA, USA) for 60, 90, and 120 min. Sperm cells were then subjected to electrophoresis to remove non-associated exogenous DNA. DNA (endogenous and exogenous) was extracted and real-time PCR was performed to quantify exogenous DNA/spermatozoa association. Each reaction was performed with 12.5 µL of Platinum SYBR Green; 0.5 µL of ROX; 5.6 µL of H2O; 5 µL of sample (1 µg); 0.7 µL of primer sense (5′-ATGGCCGACAAGCAGAAGAAC-3′) and 0.7 µL of primer anti-sense (5′-TGCCGTCCTCGATGTTGTG-3′); 500 nM each. Real-time PCR was conducted for 40 cycles of 95°C for 15 s and 64°C for 1 min. Apoptosis and oncosis analyses were evaluated by flow cytometry with 106 sperm/mL stained with 2 µL of Yo-pro (100 µM) for 20 min and with 10 µL of propidium iodide (6 µM) for 10 min at room temperature. Data were analyzed by MINITAB Realease 14 Statistical Software (Minitab, Inc., State College, PA, USA), subjected to ANOVA, and compared by Fisher's or Tukey's test (P ≤ 0.05) for transfection rate and apoptosis/oncosis, respectively. Linear regression obtained by the amplification of pEYFP-Nuc at different concentrations showed efficiency of R2 = 0.9987. The results summarized in Table 1 show that increase in length of the incubation period increases the amount of exogenous DNA associated with spermatozoa and does not affect the average of live spermatozoa in apoptosis, oncosis, and initial oncosis and/or late apoptosis groups.
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This work was supported financially by FAPESP 03/07456-8 and 03/10234-7.
