(A) The number (left) and duration (right) of mounting behavior of sexually naïve males toward ovariectomized females painted with vehicle (n = 16), non-estrus urine (NEU; n = 14) or estrus urine (EU; n = 22). EU and NEU were collected 2 and 4 days, respectively, after PMSG injection to induce estrus. Error bars, SEM; *p<0.05 (Mann–Whitney test). (B) Traces showing GCaMP2 responses of a representative cell to urine samples collected from females 1 to 5 days after PMSG injection and SEs. Arrows indicate the onset of stimulus delivery. (C) Representative images of the VNO slice response pattern to E1050 (magenta) and urine (green). Scale bar, 50 μm. (D) Bar graph showing the percentage of SE-activated VSNs (n = 86; 3 slices) that are also activated by female urine samples. (E) Venn diagrams showing the overlap between VSNs responding to SEs and those activated by EU or NEU.