Fig. 4: Conditional deletion of Tgfb1 in cumulus cells and Tgfbr2 in ampullary epithelial cells blocks NPPC expression and sperm migration in the oviduct.

a, b Knockout efficiency of TGFB1 in cumulus cells and TGFBR2 in the oviductal ampullae were detected by immunofluorescence (a) and Western blotting (b). (n = 3 mice in each group). OCCs and ampullae were isolated from superovulated mice at 13 h post-hCG treatment. Nuclei were counterstained by DAPI (blue). Scale bars represent 50 μm. c, d NPPC mRNA (c) and protein (d) levels in the ampullae isolated from mice before and after hCG injection (at 13 h post-hCG treatment). (n = 5 independent experiments in (c) and n = 3 independent experiments in (d)). e NPPC protein levels in the ampullae isolated from superovulated mice at 13 h post-hCG treatment (n = 3 independent experiments). f Representative images of the oviductal ampulla and lower isthmus isolated from mice ~3 h postcopulation (at 16 h post-hCG treatment). (n = 6-7 mice in each group). The dashed line represents the edge of the oviductal lower isthmus. Amplified views of the boxed area are shown in the lower pane. Arrow indicates spermatozoon in the oviductal ampulla. Scale bars represent 100 μm. g The number of spermatozoa in the ampullae. Oviducts were isolated from mice ~3 h postcopulation (at 16 h post-hCG treatment), and the number of spermatozoa in the ampullae were counted for each mouse. (n = 6–7 mice in each group). h Number of oocytes ovulated by Tgfb1^fl/fl, Tgfb1^cKO, Tgfbr2^fl/fl, and Tgfbr2^cKO mice (n = 6–8 mice in each group). i Representative images of the two-cell embryos. Scale bars represent 100 μm. j, k Rate of two-cell embryos (j) and the number of pups per litter (k). (n = 6–17 mice in each group). Two-cell embryos were obtained from mice at 1.5 days post coitum. β-actin was used as a loading control in (b, d, and e). Bars indicate the mean ± SD. Each data point represents a biologically independent experiment in (b–e), or represents a single mouse in (g, h, j, and k). Statistical analysis was performed by two-tailed unpaired Student’s t-test in (b, g, h, j, and k), and by one-way ANOVA Tukey test in (c–e). ns, no significance (P ≥ 0.05). *P < 0.05, **P < 0.01, and ***P < 0.001.