319 VALIDATION OF THE CARBOXYFLUORESCEIN DIACETATE ASSOCIATED WITH PROPIDIUM IODIDE FOR MURINE SPERM ACROSOME AND PLASMA MEMBRANE INTEGRITY

Abstract

The spermatozoa is an ideal vehicle for genetic modification, production of transgenic animals, as well as a biotechnological tool for sperm-mediated gene transfer. However, in order to achieve successful sperm fertilization and exogenous DNA integration, it is necessary for viable cells to remain intact, allowing the sperm to penetrate the oocyte. Fluorescent probes allow evaluation of morphological and functional characteristics of cells, which can be evaluated separately or simultaneously. Therefore, the aim of this study was to validate the simultaneous evaluation of the integrity of plasma and acrosomal membranes of murine sperm using the probes carboxyfluorescein diacetate (CF) and propidium iodide (PI). In order to validate, a standard curve was performed. Sperm were obtained from epididymis and vas deferens from CD-1 mice (8 to 16 weeks of age). Recovered samples were diluted in PBS and then divided into 2 aliquots: one prepared with fresh semen (FS) and the other submitted to Percoll gradient (45%/90%) followed by flash-freezing in liquid nitrogen and thawing (FTP) to induce acrosome damage. Samples were prepared with the following average of FS:FTP: 100:0 (T100), 50 : 50 (T50), and 0 : 100 (T0). Samples were stained using 2 μL Hoescht 33342 (40 μLmL-1 in Dulbecco’s phosphate buffered saline), 3 μL of PI (0.5 mg mL-1 in PBS), 3 μL of CF (0.46 mg mL-1 in DMSO), and were incubated for 8 min at room temperature. After staining, the samples were placed on a slide, coverslipped, and evaluated immediately by epifluorescent microscopy. The Hoescht, PI, and CF fluorescence was detected using a filter with excitation at 352, 538, and 495 nm and emission at 455, 617, and 517 nm, respectively. Approximately 200 sperm cells per slide were examined and classified based on the fluorescence emitted from each probe. Spermatozoa CF+/IP- were considered as intact membranes, CF+/PI+ as acrosome membrane intact and plasma damaged, CF-/PI+ as damaged membranes, and CF-/PI- as acrosome membrane damaged and plasma intact. Hoeschst was used as positive dye. This experiment was replicated 6 times per group, and for statistical analyses, the data of plasma and acrosomal membrane integrity (dependent variables) in the treatments T0, T50, and T100 (independent variables) were submitted to simple linear regression analysis by STATVIEW software (SAS Institute Inc., Cary, NC, USA). The CFDA/PI probes were suitable for the analysis of acrosomal and membrane status of murine sperm and showed a high determination coefficient to plasma membrane integrity (R2 = 0.81; Y = 0.5412x + 6.375) and acrosome integrity (R2 = 0.85; Y = 0.5653x + 11.653). The described protocol was efficient for the simultaneous evaluation of plasma and acrosomal membrane integrity of murine spermatozoa, proving that CFDA can be employed to access acrosomal integrity as an alternative to FITC-PSA. Financial support: FAPESP.