Supplementary Material for: Expression of toll-like receptors in the lung tissue of mouse fetuses generated by in vitro embryo culture and embryo transfer

Mouse fetuses generated by in vitro embryo culture and embryo transfer exhibit impaired lung development, altered composition of pulmonary epithelial cells associated with down regulation of several genes involved in lung development and toll-like receptor (TLR) signaling pathway. The aims of the present study were to determine the expression all TLRs, and, to examine if the expression of TLRs, along with genes involved in TLR-signaling pathway, is altered in the lung tissue of mouse fetuses generated through embryo culture and embryo transfer. Two experimental (EGs) and one control (CG) group were included in the study. Embryos cultured at 5% C02-95% air for 95 hours or less than 24 hours were transferred to pseudo-pregnant females to obtain fetuses comprising EGin vitro(n=18) and EGin vivo(n=18), respectively. Fetuses obtained from naturally-ovulating females on day-18 of pregnancy served as the CG(n=18). Western blot and immunohistochemistry were used to determine the expression of TLR proteins. The expression of transcripts encoding TLRs, and the genes involved in TLR-signaling pathway (Lbp, Pik3r1, Pik3cb, Nfkbia and Fos) was determined using qRT-PCR. While all TLRs were expressed by cells lining the bronchial/bronchiolar epithelium of lung tissues in all groups, some of the TLRs were expressed in a specific pattern. When compared to CG, the expression of transcripts encoding TLR-2, -3, -4, -5, -7, -8, -9, -12, -13, Lbp, Pik3r1, Pik3cb, Nfkbia and Fos was significantly down-regulated in both EGs. It appears that stress imposed on embryos at preimplantation stages of development is associated with down regulation of TLRs, along with some of the genes involved in TLR-signaling pathway, in the lung tissue during the perinatal period. It remains to be determined if down-regulation of TLRs, along with the genes involved in TLR signaling pathway, has any functional consequences in the adult lung tissue.