110 OSMOTIC STRESS ON THE CELLULAR ACTIN FILAMENT ORGANIZATION OF IN VITRO PRODUCED PORCINE EMBRYOS

Abstract

Disruption of the actin cytoskeleton is one of the leading causes in low survival of pig embryos after cryopreservation (Dobrinsky et al., 2000 Biol. Reprod. 62, 564–570). In this study, the effect of osmotic stress on cellular actin filament organization in porcine embryos produced in vitro was studied. Excellent quality Day 6 (fertilization = Day 0) porcine blastocysts were randomly exposed to 6 different anisosmotic sucrose solutions (75, 150, 210, 600, 1200, 2400 mOsm) for 10 min. Embryos were then returned to embryo culture medium (NCSU-23) after washing with NCSU-23, and cultured under 38.5°C, 5% CO₂ in air with maximal humidity for them to recover. Blastocysts cultured in NCSU-23 medium (280 mOsm) served as a control for embryos with intact actin filament organization. Blastocysts treated with 7.5 μg mL⁻¹ cytochalasin-b for 60 min served as a control for embryos with F-actin depolymerization. Eighteen hours post-anisosmotic treatments, all blastoysts were fixed in 3.7% paraformaldehyde in PBS for 60 min and stored in PBS with 0.1% Triton X-100 and 0.2% sodium azide at 4°C. Staining of actin filaments was performed according to procedures described earlier (Wang et al., 1999 Biol. Reprod. 60, 1020–1028). Embryos were blocked in PBS with 20 mg mL⁻¹ BSA and 150 mM glycine for 30 min. After being washed in PBS with 0.1% Tween 20 for 60 min, embryos were stained with 10 U mL⁻¹ Alexa Fluor 488 phalloidin in PBS with 0.1% Tween 20 at 38.5°C for 60 min, and then washed twice in PBS with 0.1% Tween 20 for 60 min each. The status of actin filaments in embryonic cells was examined by confocal microscopy. Integrity of cellular actin filaments was classified as either intact or disrupted according to the distribution within embryonic cells. Blastocysts were then classified according to the status of actin filaments in embryonic cells. Data were analyzed using logistic regression. Results from 7 replicates are displayed in Table 1. There was a significant relationship between osmotic treatment levels and the probability of blastocysts with disrupted cellular actin filaments (P < 0.0001). These data support the hypothesis that porcine embryos are very sensitive to osmotic changes. Ongoing experiments will assess the extent of actin disruption required to significantly reduce developmental competence of pig blastocysts. This study was supported by Monsanto Company.

Table 1 
Cellular actin filament integrity of in vitro produced porcine blastocysts after being treated with sucrose solutions with different osmolalities (mOsm)