Fig. 3: OCC-stimulated NPPC promotes sperm migration from the isthmic reservoir to the ampulla.

a, b OCC and TGFB1-induced NPPC mRNA (a) and protein (b) levels were blocked by SB431542 and SD208. (n = 5 independent experiments in (a) and n = 3 independent experiments in (b). The ampullae were isolated from superovulated mice at 11 h post-hCG (without OCCs in the ampullae), and OCCs were collected from the ampullae of superovulated mice at 13 h post-hCG treatment. The ampullae were cultured with OCCs (3 OCCs/μl), TGFB1 (5 ng/ml), SB431542 (SB, 5 μM), and/or SD208 (SD, 1 μM) in a 50 μl drop for 3 h, and NPPC mRNA and protein levels were determined at the end of the culture. β-actin was used as a loading control. Each data point represents a biologically independent experiment. c Representative frames from the time-lapse imaging of the oviductal lower isthmus (left panel) and representative images of the ampulla at the end of the time-lapse imaging (right panel). (n = 6 mice in each group). The dashed line represents the edge of the lower isthmus. Oviducts were isolated from mice at the stated time. Amplified views of the boxed area are shown on the right-hand side. Arrow indicates spermatozoon in the oviductal ampullae. Scale bars represent 100 μm. Posto, postovulatory oviduct; Preo, preovulatory oviduct. d The number of spermatozoa in the ampullae at the end of the time-lapse imaging (n = 6 mice in each group). Each data point represents a single mouse. Statistical analysis was performed by one-way ANOVA Tukey test. Bars indicate the mean ± SD. ns, no significance (P ≥ 0.05). *P < 0.05 and ***P < 0.001.