138 LIVER RECEPTOR HOMOLOG 1 INFLUENCES BLASTOCYST HATCHING IN PORCINE

Abstract

Liver receptor homolog 1 (LRH1, NR5A2) belongs to the orphan nuclear receptor superfamily and has diverse functions in development, differentiation, metabolism, and cell death. The LRH1 gene is expressed in embryonic stem cells and regulates the expression of OCT4, which is the key factor required to maintain pluripotency. However, the function of LRH1 in early porcine embryo development is still unknown. In the present study, we examined the localization and mRNA level of LRH1 in porcine parthenotes by immunofluorescence and real-time RT-qPCR. To explore the function of LRH1, the embryos were treated with the specific antagonist of LRH1, 505601, which is a cell-permeable pyrazolylbiphenylethanone compound that targets LRH-1/NR5A2 ligand binding domain (LBD) via direct affinity interaction, preventing LRH-1 from assuming an active conformation. Every group contained 20 embryos. The results showed that the immunofluorescence signal of LRH1 was only observed in nuclei of blastocyst cells. Inhibition of LRH1 by antagonist significantly (P < 0.05) reduced the blastocyst rate in the 50 µM (37.30 ± 3.67%) and 100 µM (38.02 ± 5.12%) treatment groups compared with the control group (53.43 ± 3.67%). The hatching rate of blastocysts was also reduced (P < 0.01) by 50 µM (12.66 ± 3.13%) and 100 µM (20.10 ± 2.81%) antagonist compared with control (40.59 ± 0.59%), which resulted from the decreased (P < 0.05) expression of hatching-related genes, such as FN1, ITGA5, and COX2. Inhibition of LRH1 also increased (P < 0.05) the number of apoptotic cells in blastocysts. Moreover, inhibition enhanced expression of apoptotic genes, BAX and CASP3, and reduced the anti-apoptosis gene BCL2 (P < 0.05). Messenger RNA and protein level of OCT4 were sharply decreased in blastocysts after LRH1 inhibition. All data were analysed with a one-way ANOVA, and differences between treatment groups were assessed by the least significant difference (LSD) test using SPSS software (SPSS Inc., Chicago, IL, USA). In conclusion, LRH1 affects blastocyst formation and hatching by regulating OCT4 expression and cell apoptosis.