273 APOPTOSIS EVALUATION OF IN VITRO-PRODUCED PIG EMBRYOS (PARTIAL RESULT)

Abstract

Apoptosis is a physiological event involved with death and tissue replication, fulfilling an important function of tissue organizations during embryogenesis. This mechanism occurs in in vivo as well as in vitro pre-implantation embryos, but most frequently in the latter. The transcriptional activation of pig embryos occurs at the four-cell stage, which is the longer stage during the pre-implantation period. This stage is characterized by embryonic developmental blockage that decreases the production rates (embryos loss). The aim of this study was to evaluate a correlation between apoptosis mechanism and developmental blockage of IVP porcine embryos. Immature oocytes after IVM/IVF were submitted to IVC in PZM-1 medium containing BSA 3 mg/mL at 38.5°C, 5% CO₂ in air and high humidified atmosphere. The embryo development was analyzed at 96 h of cultute (Day 4) in order to verify cleavage rate and blockage (4 cells) and non-blockage (e8 cells) embryo rates. Out of 625 grade I, II, and III oocytes submitted to IVP, 70.3 ± 5.2% (430/625) cleaved from which 27.1 ± 10.3% (166/625) were blocked and 43 ± 10.8% (264/625) were non-blocked. Blocked and non-blocked embryos were assessed to evaluate apoptosis rates. Qualitative assays of embryo cells were achieved with two different DNA stains: YOPRO-1 (Molecular Probe^®; Invitrogen Brasil, Ltd., Sao Paulo, Brazil), permeable though plasma membrane in the early stage of apoptosis, and TUNEL (Roche^®; Amersham Biosciences, Sao Paulo, Brazil), which detects DNA fragmentation in the last stages of apoptosis. The embryos were stained with 0.1 µM YOPRO-1/mL PBS, incubated 15 min at 38°C, 5% CO₂ in air and high humidified atmosphere, and immediately observed by means of confocal microscopy. For the TUNEL assay, embryos were fixed in 4% paraformaldehyde solution (w/v) in PBS for 1 h at room temperature, and incubated in permeabilization solution [0.5% (v/v) Triton X-100, 0.1% (w/v) sodium citrate in PBS] for 2 h. For positive control, samples were treated with DNase-I at 37°C for 1 h. The negative control and experimental samples were incubated with buffer solution under the same conditions. The positive control and experimental samples were incubated with enzymatic and stain solution (FITC) at 37°C for 1 h; the negative control was incubated with only enzymatic solution. The embryos were stained with Hoechst 33342 (5 µ/mL) and observed by means of fluorescence microscopy. Blocked embryos showed more apoptosis (66% and 40% to YOPRO-1 and TUNEL, respectively) than non-blocked embryos (25% and 0% to YOPRO-1 and TUNEL, respectively). In conclusion, the developmentally blocked embryos suffered more apoptosis, although morphologic apoptosis assays (light and electronic microscopic) must be performed to confirm this finding.

This work was supported by FAPESP 04/01252-4.