Involvement of TLR7 and TLR8 in Conceptus Development and Establishment of Pregnancy in Sheep

Toll-like receptors (TLRs) belong to the innate immune system and regulate inflammatory events that impact mammalian reproduction. In Study 1, we demonstrated that abundance of ovine TLR 1-9 mRNAs in the uterus differs due to reproductive status (TLR2-3,TLR7-8) and day of the oestrous cycle and pregnancy (TLR1-3,TLR5-7,TLR9). Expression of TLR7 and TLR8 proteins was localized primarily to uterine epithelia and stroma and regulated in a temporal manner. In Study 2, we determined that ovine conceptuses express TLR7 and TLR8 on all days studied and that expression of the envelope protein of ovine endogenous retrovirus (enJSRV-Env) declined in conceptus trophectoderm from Day 13 to Day 16 of pregnancy. In Study 3, loss of function experiments were conducted in vivo using morpholino antisense oligonucleotides (MAO) injected into the uterine lumen to block synthesis of TLR7 and TLR8 proteins, individually and jointly. Conceptuses were recovered on Day 16 to assess their morphology. MAO-treated conceptuses were developmentally retarded, produced less interferon-tau (IFNT) and had fewer binucleate cells (BNC) compared to MAO-Controls. Moreover, expression of enJSRV-Env mRNA in MAO-TLR7 conceptuses was greater than for MAO-Control and MAO-TLR8 conceptuses, but similar to MAO-TLR7/8 conceptuses. Results of this study indicated differences in TLR1-9 expression due to reproductive status and day of the oestrous cycle and pregnancy. Also, TLR7 and TLR8 influence development, enJSRV-Env abundance, secretion of IFNT and formation of BNC by conceptuses. These findings support our hypothesis that TLR7 and TLR8 mediate pathways whereby enJSRV-Env regulates key peri-implantation events in conceptus development and differentiated functions of trophectoderm cells.