127 BINDING RETINOID RECEPTORS BY SPECIFIC AGONISTS AFFECTS THE BOVINE BLASTOCYST DEVELOPMENT IN VITRO

Abstract

Production of embryos in vitro with improved inner cell mass (ICM) and high ICM per total cell rate is a major objective in reproductive biotechnology. Exogenous all-trans retinoic acid (ATRA), a vitamin A metabolite, and endogenous retinoid regulate development and differentiation during bovine morula to blastocyst transition in vitro. ATRA binds to retinoic acid-receptor (RAR), and the ATRA isomere 9-cis-retinoic acid (9-cis-RA) binds to both RAR and the retinoid X receptor (RXR). The unspecific binding of 9-cis-RA to receptors makes it difficult to study RXR transactivation. Therefore, in this work we studied blastocyst development and cell counts by using a specific synthetic RXR agonist [LG100268 LG; a gift of Ligand Laboratories] as opossed to the effect exerted by ATRA upon RAR binding. Cumulus-oocyte complexes from slaughterhouse ovaries were matured and fertilized in vitro. Presumptive zygotes were cultured in B2 medium with Vero cells until 139 h post-insemination (Day 6), the time at which embryos [morulae (e90%) + early blastocysts] underwent treatments for 48 h in 400 ¼L of SOFaaci + 5% FCS. Data (5 replicates per experiment) were analyzed by CATMOD for effects, processed by GLM and Duncan's test, and expressed as LSM ± SE (a,b,c P d 0.05). After a LG dose-response experiment (n = 480 morulae), blastocysts rates from LG 1 ¼M on Day 7 were higher than LG 10 ¼M, LG 0.1 ¼M, and LG 0 ¼M (Day 7: 42.8 ± 4.1 vs. 34.4 ± 3.7, 36.8 ± 3.7, and 32.4 ± 3.7, respectively). On Day 8, LG 1 ¼M also yielded more blastocysts than LG 0.1 ¼M (50 ± 4.2 vs. 44.4 ± 3.7, respectively). By differential cell counting (n = 113 blastocysts), hatched blastocysts with LG 10 ¼M showed proliferation in the ICM, while trophectoderm (TE) cells decreased conversely to LG concentration. These effects were not obvious in expanded blastocysts. In a subsequent experiment (n = 340 morulae), ATRA led to blastocysts rates on Day 8 that were higher than negative, untreated controls, but not different from LG 1 ¼M (42.4 ± 2.4 vs. 33.1 ± 2.0 and 36.0 ± 2.4, respectively). ATRA and LG 1 increased TE in expanded blastocysts (n = 42) (102 ± 13.2 and 96.23 ± 13.2, respectively vs. 72.8 ± 10.9 in the untreated group) but not in their hatched counterparts (n = 44). There were no differences in the ICM; but percentages of ICM per total cells were higher in hatched blastocysts cultured with ATRA than in expanded LG 1 ¼M blastocysts and expanded controls (39.5 ± 5.5 vs. 24.2 ± 5.7, and 20.9 ± 4.7, respectively). Manipulation of retinoid receptor-specific pathways make it possible to control blastocyst development and differentiation, leading to embryos of improved quality and viability. Work is in progress to analyze gene expression in these blastocysts.

This work was supported by grant MCYT, project AGL-2005-04479.