Figure 2. Genetic Disruption of Efcab9 Compromises Male Fertility, Sperm Motility, and CatSper Domain.

(A) Pregnancy rate of Efcab9^−/− compared with Efcab9^+/− males over 2 months when housed together with WT females.

(B) In vitro fertilization rate of Efcab9^+/− (circles, 64.2% ± 11.0%) and Efcab9^−/− (diamonds, 23.8% ± 14.7%) sperm with cumulus oocyte complex. Each dotted line indicates the results from each paired replication (n = 3). *p < 0.05.

(C) Flagellar bending of mouse sperm. Red lines indicate the curvature of the sperm head (hook). Pro-hook (left) and anti-hook (right) bends refer to the primary flagellar bend in the same or the opposite direction to the hook of the head, respectively. </p/> (D) Flagellar waveform of Efcab9^+/− (left) and Efcab9^−/− (right) sperm cells. Movies recorded at 200 fps from the head-tethered cells to glass coverslips before (top, 0 min) and after (bottom, 90 min) incubating under capacitation conditions. Overlays of flagellar traces from two beat cycles are time-coded in color. Pro-hook and anti-hook directions are marked with filled and empty arrows, respectively.

(E) The maximal bending angle of the midpiece (α-angle) in primary anti-hook curvature was measured from Efcab9^+/− (0 min, 52.1° ± 2.7°; 90 min, 90.1° ± 2.3°) and Efcab9^−/− (0 min, 40.0° ± 2.2°; 90 min, 33.8° ± 2.1°) sperm, and tail parallel to head is considered to 0°. Data are represented as mean ± SEM. n = 50 for each group. ***p < 0.001.

(F) Beat frequency before (0 min, Efcab9^+/−, 13.9 ± 0.4 Hz; EFCAB9^−/−, 23.5 ± 0.9 Hz) and after (90 min, Efcab9^+/−, 8.19 ± 0.27 Hz; Efcab9^−/−, 26.2 ± 1.2 Hz) incubating under capacitation conditions is shown in boxplot, indicating quartile distribution of individual data points. Median ± SEM, n = 50 each group (Efcab9^+/−, circles; Efcab9^−/−, diamonds). ***p < 0.001.

(G) Flagellar bending patterns of sperm from Efcab9^+/− (left) and Efcab9^−/− (middle and right) mice. Flagellar waveform were examined and classified into those bent in both directions (green) or in a fixed direction (blue). Sperm cells that beat in one direction are further divided into pro-hook (white) or anti-hook (cyan). Sperm numbers in the pie charts are from three independent experiments.

(H) Efcab9^−/− spermatozoa have normal sperm morphology but display discontinuous CatSper domains. Overlay of confocal images and the corresponding DIC images of immunostained WT (top) and Efcab9^−/− (bottom) sperm cells by α-EFCAB9 (left) and α-CatSper1 (right). SIM images at the right side of the CatSper1 confocal images are from the inset. Arrowhead indicates annulus, the junction between the midpiece and principal piece of the sperm tail.

See also Figures S3 and S4 and Video S1.