Fig 1.
CFTR is prominently reduced in the epididymis and vas deferens of Slc9a3-/- mice.
(A, B, C) CFTR expression in the caput (A) and cauda (B) epididymis and vas deferens (C) of 2-month-old Slc9a3-/- mice compared with that in WT mice was assessed by western blotting. In each panel, the upper image shows the CFTR expression (arrow indicates the fully glycosylated form) and the lower image shows the β-actin expression, which was used as a loading control. Each group comprised three WT and Slc9a3-/- mice. (D) Western blotting results were quantified through a morphometric analysis. For each organ, the CFTR intensity was normalized to that of β-actin expression, and the mean intensity in WT mice was used as a reference to calculate the fold change in expression in Slc9a3-/- mice. Each bar represents the mean ±SEM; n = 3 per genotype. *Significant difference compared with WT mice (***p < 0.0001, analyzed using the Student’s t test).
Table 1.
Comparison of fertile capability between wild-type, heterozygous, and homozygous Slc9a3 knockout mice.
Fig 2.
SLC9A3 expression in the epithelium of male excurrent ducts of 2-month-old WT mice.
Localization of SLC9A3 (green, middle panel) and pan-cytokeratin (red, upper panel) in the efferent ducts (A); caput (B), corpus (C), and cauda (D) epididymis and vas deferens (E) of WT mice was detected by performing immunofluorescence double labelling with specific primary antibodies. Pan-cytokeratin was used as a specific marker of differentiated epithelial cells. The nucleus was stained with DAPI (blue) and is displayed in merged images (lower panel). The insets show higher magnification of the indicated boxed areas; scale bar = 20 μm.
Fig 3.
Gross morphology of the reproductive organs of infertile Slc9a3-/- male mice.
(A) Intact appearance of overall reproductive organs, including the seminal vesicle (SV), bladder (Bla), vas deferens (VD), testis (T), and epididymis (Epi), of 2-month-old WT and Slc9a3 deficiency mice. (B, C, and D) Enlarged images of the testis (B), epididymis (C), and vas deferens (D). Statistics show the ratio of the normalized organ weight (mg) to body weight (g). Each bar represents the mean ± SEM; n = 10 per genotype. *Significant difference compared with WT mice (*p < 0.05 and ***p < 0.0001, analyzed using the Student’s t test).
Fig 4.
Loss of SLC9A3 induces testicular atrophy.
Testicular histology was assessed by H&E staining of testis sections of 6-month-old WT mice (A, B) and Slc9a3-/- mice of different ages (C–H). (A, B) Representative images showing the intact architecture of the seminiferous tubules and interstitial tissue in the testes of WT mice. (C–H) H&E staining of the testes of 2- (C, D), 4- (E, F), and 6–month-old (G, H) Slc9a3-deficient mice. Vacuolation of seminiferous epithelia was observed in the seminiferous tubules of Slc9a3-/- mice (asterisk). The dashed line marks the intact lumen of the seminiferous tubules. The lower panel is the higher magnified images of the area indicated using a black dashed line in the upper panel; scale bar = 20 μm. Abbreviations: Sg, spermatogonia; Sc, spermatocyte; Sd, spermatid; Sz, spermatozoa.
Fig 5.
The lumen of the efferent ductules of 4-month-old Slc9a3-/- males is dilated and calcified.
Comparison of the efferent ducts of 4-month-old WT (A, B) and Slc9a3-/- (C, D) mice according to H&E staining of the epididymal sections. (A, C) Each representation is a montage assembled by a sequence of film taken with a ×10 objective on the bright-field microscope. The circled region with black solid line in the upper part of the montage is the efferent ductule, and the lower part of the montage is the epididymis. (B, D) Enlarged images from the areas boxed by a red dashed box in the upper panel. The widest distance from one side to the other side of the lumen is marked by double-headed arrows, and cilia on the epithelium are indicated by black arrows. Calcification (red arrow) was observed in some dilated ducts. Scale bar = 200 μm (A, B) and 20 μm (C, D)
Fig 6.
Absence of spermatozoa in the caput epididymis of adult Slc9a3-/- mice.
Histological pathology of the caput epididymis of 6-month-old WT (A, B) and 2- (C, D), 4- (E, F), and 6-month-old (G, H) Slc9a3-/- mice was assessed by H&E staining. (A, C, E, G) Each montage, which contains a series of images obtained with a ×10 objective, displays the general morphology of the caput epididymis section. (B, D, F, H) Enlarged view is marked by a black dashed line in the upper panel. Spermatozoa are marked by arrowheads. Abnormal secretions (arrow) observed in the ducts of the caput epididymis of Slc9a3-/- male mice. Scale bar = 200 μm (A, C, E, G) and 20 μm (B, D, F, H).
Fig 7.
Slc9a3-/- cauda epididymis is filled with abundant abnormal secretions instead of spermatozoa.
H&E-stained sections of the cauda epididymis of 6 month-old WT (A, B) and Slc9a3–/–males aged 2 months (C, D), 4 months (E, F), and 6 months (G, H). (A, C, E, G) The low magnification overview is a montage assembled by an array of films continuously taken with a ×10 objective on a bright microscope. (B, D, F, H) Magnified view of the area indicated by a dashed box in the upper panel. Spermatozoa are indicated by arrowheads, and abnormal heterogeneous secretions are marked by arrows, a dashed arrow, and a green arrow. The white lines are sectioning artefacts. Scale bar = 200 μm (A, C, E, G) and 20 μm (B, D, F, H).
Fig 8.
Aberrant secretions within the lumen of the vas deferens in Slc9a3-/- males.
Histology of the vas deferens of 6-month-old WT (A, B) and 2- (C, D), 4- (E, F), and 6-month-old (G, H) was examined by H&E-stained longitudinal sections. (A, C, E, G) General morphology at low magnification. (B, D, F, H) Micrograph showing a magnified view of the boxed area indicated in the upper panel. The arrowhead indicates spermatozoa. The arrow indicates abnormal heterogeneous secretions. The wrinkles on sections are sectioning artefacts. Scale bar = 200 μm (A, C, E, G) and 20 μm (B, D, F, H).
Fig 9.
Ultrastructural defects of the epithelium in Slc9a3–/–epididymis and vas deferens.
An electron micrograph (cross-section) showing the ultrastructure of the epithelia of the excurrent ducts of 2-month-old WT (upper panel) and Slc9a3-/- male mice (lower panel). (A, B) Caput epididymis. The arrow indicates the absence of stereocilia on the caput epithelium. The black dashed line marks the boundary between the lumen and the edge of stereocilia on the epithelium. (C, D) Cauda epididymis. The white arrowhead indicates the disturbed and decreased number of stereocilia. The light blue arrow indicates the vesicles in the cauda epithelium. (E, F) Vas deferens. The asterisk indicates abnormal secretory particles and the star indicates smooth endoplasmic reticulum-like material in the particles (enlarged image in S2 Fig). The magnification for each image is slightly different, with 2000× magnification used for A, B, and E and 2500× magnification used for C, D, and F. Scale bar = 5 μm. Abbreviations: B, basal cell; M, mitochondria; N, nucleus; S, stereocilia; Sz, spermatozoa.
Fig 10.
Proposed working model of the role of SLC9A3 in the pathogenesis of the male reproductive system.
The upper right diagram depicts the possible cooperation between SLC9A3 and CFTR in regulating luminal homeostasis. The lower schematic diagram summarizes the pathological features of Slc9a3-/- male mice. The color gradient in the epididymis represents the amount of abnormal heterogeneous secretions progressively increasing from the caput (light purple) to the cauda (dense purple) epididymis.