Retinoid receptor-specific agonists regulate bovine in vitro early embryonic development, differentiation and expression of genes related to cell cycle arrest and apoptosis

Abstract

A major goal in reproductive biotechnology is the identification of pathways that regulate early embryonic development and the allocation of cells to the inner cell mass (ICM) and trophectoderm (TE). Retinoids regulate the development and differentiation of the bovine blastocyst in vitro, although the involvement of the retinoid X receptors (RXRs) remains to be clarified. This paper compares the effect of a synthetic RXR agonist (LG100268; LG) with that of the retinoic acid receptor (RAR) agonist all-trans retinoic acid (ATRA) on blastulation. In vitro-produced morulae were treated for 48 h with LG (0.1 mM, 1 mM and 10 mM), ATRA 0.7 mM, or no additives. Treatment with ATRA did not increase the rate of development; however, the LG 0.1 mM treatment increased both the blastocyst development and hatching rate. Cell numbers increased in the ICM with LG 10 mM, while a dosedependent reduction was observed in the TE in the presence of LG. Gene expression levels of p53 and p66 did not vary with LG but increased with ATRA. Both LG and ATRA activated bax, a pro-apoptotic gene and H2A.Z, a cell cycle-related gene. The above effects suggest the existence of active p53-dependent and -independent apoptotic pathways for ATRA and LG, respectively, in the bovine embryo. The expression of p53 and H2A.Z showed a strong, positive correlation (r = 0.93; p < 0.0001) in all experimental groups; both proteins are linked through the cell cycle. Agonists of RXR could be used to control blastocyst development and differentiation. #

127 Development and Quality of Bovine Morulae Cultured in Serum-Free Medium With Retinoic Receptor Specific Agonists

“…Treatment with LG for 24 h did not affect cell counts, which is in contrast with the finding of a significant reduction in the number of cells allocated to the TE after 48 h culture in the presence of 10 µm LG reported by Rodríguez et al (2007). In the same experiment, it was shown that the effects of LG on blastocyst development and differentiation were dose dependent.…”

“…It has been shown previously that both endogenous and exogenous retinoids play a role in apoptosis (Rodríguez et al 2007). In the present study, there were essential differences in the induction of apoptosis depending on the retinoid ligand:…”

“…However, in a recent paper (Rodríguez et al 2007), blastocyst rates obtained after 48 h treatment of morulae with ATRA-containing medium were similar to those obtained in the control (no additives) group, but lower than those seen in embryos cultured with 0.1 µm LG. The lack of effect of LG in culture for 24 h contrasts with previous results, where embryos cultured in the presence of 0.1 µm LG for 48 h showed improved blastocyst development and had larger diameters (Rodríguez et al 2007).…”

“…In addition, ATRA treatment tended to result in a reduced total NI compared with LG. The effects of ATRA on apoptosis were opposite to those observed when endogenous RA production is inhibited in blastocysts (Rodríguez et al 2007), suggesting that retinoids regulate apoptosis in the blastocyst. In cultured mouse blastocysts, the antiproliferative and pro-apoptotic effects of 10 µm ATRA were mainly targeted to the ICM (Huang et al 2005).…”

“…However, we have demonstrated that long exposure (48 h) to 0.1 µm LG improves blastocyst development (Rodríguez et al 2007) and that short exposure (24 h) to 0.7 µm ATRA increases cell numbers in the inner cell mass (ICM) and trophectoderm (TE) without changing the ICM/TE cell ratio (Rodríguez et al 2006). Classically, the quality of the IVP bovine blastocyst has been associated with the amount and proper allocation of embryonic cells to the ICM and TE (Van Soom et al 1996), as well as their sensitivity to cryopreservation (Imai et al 2002;Lonergan et al 2003aLonergan et al , 2003bRizos et al 2003).…”