127 DEVELOPMENT AND QUALITY OF BOVINE MORULAE CULTURED IN SERUM-FREE MEDIUM WITH RETINOIC RECEPTOR SPECIFIC AGONISTS

Abstract

In the cell, all-trans retinoic acid (ATRA), a vitamin A metabolite, binds to retinoic acid-receptor (RAR), whereas the ATRA isomere 9-cis-retinoic acid (9-cis-RA) binds to both RAR and the retinoid X receptor (RXR). Synthetic compounds such as LG100268 (LG; Ligand Laboratories) are highly specific to bind RXR, which allows to differentially study the RAR and RXR pathways. In previous work morulae treated with LG for 48 h showed to improve blastocyst development and to activate pro-apoptotic genes (in press), whereas ATRA for 24 h increased cell numbers in the inner cell mass (ICM) and the trophectoderm (TE) (Rodríguez et al. 2006 Hum. Reprod. 21, 2149–2157). However, LG and ATRA were never both compared for 24 in medium with BSA, which is thought to be more appropriate to produce embryos for cryopreservation than serum-containing medium. In this work we analyze development, quality, and viability of morulae cultured with RAR and RXR agonists. Cumulus–oocyte complexes from slaughterhouse ovaries were matured and fertilized in vitro. Presumptive zygotes were cultured in synthetic oviduct fluid (SOF) +3 gL^–1 BSA. On day 6, morulae were treated for 24 h with ATRA 0.7 µm, LG 0.1 µm, or no additives. Blastocyst development was monitored up to day 8. Differential cell counts were made on hatched blastocysts on days 7 and 8. Apoptosis and necrosis (TUNEL + nuclear histology) were made on day 8 expanded and hatched blastocysts. Data were analyzed by GLM and Duncan's test, expressed as LSM ± SE, and development rates were expressed as percentages of cultured morulae (replicates [R] = 14 for development; R = 9 for cell counts; R = 4 for apoptosis; n = 1647 morulae). ATRA yielded more blastocysts on day 8 than LG and controls (72.2 ± 2.2 v. 60.0 ± 2.3 and 65.6 ± 2.4, respectively; P < 0.02), and more expanded blastocysts than LG (48.6 ± 2.3 v. 36.6 ± 2.4; P < 0.02), but no more than controls (43.5 ± 2.5). Day-7 and day-8 hatched blastocysts cultured with ATRA showed more total cells than day-7 controls (163.5 ± 8.0 and 161.5 ± 5.4 v. 137.7 ± 8.9, respectively; P < 0.05). However, in the presence of ATRA, day-8 blastocysts showed a strong cell reduction in the inner cell mass (ICM), whereas their day-7 counterparts conserved ICM/total cells proportions comparable to day-7 controls (11.0 ± 1.2 v. 19.7 ± 1.7 and 20.6 ± 1.9, respectively; P < 0.03). The LG increased apoptotic index (AI) and necrotic index (NI) in the ICM (AI: 14.5 ± 2.4 v. 6.4 ± 1.5 and 6.4 ± 1.4; NI: 5.0 ± 1.2 v. 0.9 ± 0.8 and 1.6 ± 0.7; for LG, ATRA, and controls, respectively; P < 0.02). Embryos produced with ATRA showed improved development and cell distribution without increasing apoptosis and necrosis. Vitrification of excellent day-7 and day-8 blastocysts is in course to evaluate cryosurvival and further embryo transfer to determine full developmental competence.

Grant Support: MEC, project AGL2005-04479. M. Muñoz is sponsored by FICYT.