191 ALLEVIATION OF THE TWO-CELL BLOCK OF ICR, ddY, AND AKR MOUSE EMBRYOS BY BOVINE SERUM ALBUMIN

Abstract

Bovine serum albumin (BSA) is an embryotrophic macromolecule used in embryo culture media and improves embryo development in vitro. However, when 1-cell embryos from some strains of mouse were cultured in traditional medium, even with BSA, developmental arrest occurred at the 2-cell stage, termed '2-cell block'. The developmental block is known to be alleviated by adding EDTA to the medium for ICR and ddY strains, and deleting phosphate from the medium for the AKR strain. Recently, our preliminary experiments revealed that the 2-cell block is relieved by adding deionized BSA (d-BSA) to the medium for the ICR strain. Thus, in the present study, we investigated whether d-BSA could rescue the embryos from ICR, ddY, and AKR strains from the 2-cell block. Fertilized 1-cell embryos were collected 20 h post-hCG from superovulated ICR, ddY, and AKR females (8-week-old) that had been mated with the ICR strain of males. Stock solutions (15%) of commercially available fraction V BSA, ovalbumin (ova), and γ-globulin (γG) were deionized over a mixed-bed ion adsorption resin. Embryos were cultured in EDTA-depleted KSOM medium with or without these deionized or non-deionized proteins at 37°C under 5% CO₂ in air for 4 days. Experiments were done in at least 3 replicates, and the statistical analyses of the data were done by ANOVA and Fisher's PLDS test. To observe the distribution of BSA in the embryos from the 1-cell to the blastocyst stages, immunofluorescence study was performed using anti-BSA antibody with a laser confocal microscope. The developmental rates to the 4-cell stage of 1-cell embryos cultured in medium without (control group) or with BSA at 0.3% in ICR and 0.6% in ddY and AKR (BSA group) were very low (ICR: 10% (4/38) and 37% (17/47); ddY: 9% (7/73) and 23% (9/37); AKR: 0% (0/60) and 0% (18/30), respectively). However, when embryos were cultured with d-BSA at 0.3% in ICR and 0.6% in ddY and AKR, the rates to the 4-cell stage significantly increased (ICR: 91% (51/56), ddY: 82% (61/76), AKR: 82% (50/60) vs. control group or BSA group: P < 0.05), and development to the blastocyst stage was observed (ICR: 79% (44/56), ddY: 65% (47/76), AKR: 63% (38/60)). When ICR embryos were cultured with 0.3% deionized-ova or deionized-³G, no significant increase was observed in developmental rates to the 4-cell stage (25% (10/40) and 24% (10/42), respectively). We next examined the critical culture period for the beneficial effects of d-BSA and intracellular distribution of BSA using ddY mouse embryos. It was found that exposure to d-BSA during the late 1-cell (24 h post-hCG) and early 2-cell stages (42 h post-hCG) promoted the development beyond the 2-cell stage. The distribution of BSA in the cytoplasm of embryos at any stage was observed. Interestingly, BSA localized in the nuclei of embryos during the late 1-cell and early 2-cell stages. In conclusion, our results suggest that BSA itself has a potential to remove the 2-cell block in ICR, ddY, and AKR strains. In addition, nuclear localization of BSA may play a key role in regulating the development beyond the 2-cell stage in the mouse embryos.