Identification of pig olfactory receptors responding to androstenone and the key structure determinant

Olfactory receptors are members of the transmembrane G protein-coupled receptor superfamily, playing crucial role in odor recognition which further mediates crucial biological processes in mammals. In sows, androstenone can trigger sexsual behaviors through olfaction, but the underlying mechanism remains to be explored. To identify the olfactory receptor responding to androstenone, we adapted the high-throughput RNAseq strategy to screen the altered genes upon androstenone treatment in olfactory epithelium of pig, yielding 1397 downregulated genes. Of which, 15 OR genes and 49 ORs-like genes are candidate androstenone responsive genes, and 6 ORs (OR2D2, OR7D4, OR8D1, OR8D2, OR10Z1 and OR7D4) were proved are responsible to androstenone mediated olfaction in vitro. Among the 6 ORs, pig OR7D4 has the highest level of androstenone response. To further find the structural determinant, we performed ligand-binding cavity analysis on pig OR7D4 with androstenone, predicted seven potential structural sites and further confirmed F178 and T203 are the key sites for recognizing androstenone. Nevertheless, the natural non-synonymous mutation M133V (rs696400829) of pig OR7D4 was proved can significantly impair the respondence to androstenone. This is the first time identified the ORs responding to androstenone in pig and the key structural determinant of pig OR7D4, which highlights the significance to investigate the role of OR7D4 on pig reproduction performance in future. Overall design: We screened for olfactory receptor genes that may respond to androstenone in the pig genome based on transcriptomic approach previously applied to species such as mice. Four sows which were offspring of Landrace (?) and Rongchang pig (?), aged 8 months, were divided into androstenone-treatment group and control group with two in each group. In the androstenone-treatment group. The Boarmate, which mainly contains androstenone, was sprayed on the nasal cavity before slaughter, while the control group received no solution treatment before slaughter. After 30 mins, the sows were sacrificed, olfactory epithelium from left and right side were collected from each sow. Samples were used for preparation of RNA-seq libraries, the candidate olfactory receptor genes responding to androstenone were screened via the RNA-seq data.