Fig. 4. C11orf94 deficiency causes normozoospermic infertility independent of SPPL2c activity.

(A) Male Wt or C11orf94-deficient (C11 KO) mice were bred with Wt females and mean litter sizes were recorded. n = 3 (Wt)/5 (C11 KO). (B) Analysis of normalized testis weight. n = 10. (C) Testis cryosections were subjected to H&E staining. Scale bars, 25 μm. (D) Analysis of sperm counts of Wt and C11 KO mice. n = 7. (E) Epididymal spermatozoa were fixed, and nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI), while acrosomes were stained with PNA-FITC. Scale bars, 5 μm. (F) Motility parameters of sperm cells were measured by CASA. VAP, average path velocity; VSL, straight line velocity; VCL, curvilinear velocity. n = 3. (G) Tail beat frequency of epididymal spermatozoa was analyzed upon addition of either HS buffer or HS supplemented with HCO₃⁻ (HSB). N = 3, n = 19 sperm per conditions. (H) Fertilization capacity of sperm cells from either Wt or C11 KO mice incubated with Wt oocytes with or without ZP was analyzed by IVF. N = 2, n = 3. (I) Testis protein levels of PLN and Stx8 were detected by immunoblotting. (J) Quantification of Stx8 levels from (I). N = 3, n = 9. (K) Quantification of PLN levels from (I). N = 2, n = 6. (L) SPPL2c protein levels were analyzed in homogenates from Wt or C11 KO testes by Western blotting. (M) Quantification from (L). n = 9. (N) Wt or C11orf94/SPPL2c double-deficient (dKO) males were bred with Wt females, and mean litter sizes were recorded. n = 3. In all cases, unpaired two-tailed Student’s t tests were performed. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.