Loss of SLC9A3 decreases CFTR protein and causes obstructed azoospermia in mice

<div><p>Mutations in the cystic fibrosis transmembrane conductance regulator (<i>CFTR</i>) gene cause cystic fibrosis (CF) and are associated with congenital bilateral absence of the vas deferens (CBAVD), which is the major cause of infertility in male patients with CF. However, most Taiwanese patients with CBAVD do not carry major <i>CFTR</i> mutations. Some patients have a single copy deletion of the solute carrier family 9 isoform 3 (<i>SLC9A3</i>) gene. SLC9A3 is a Na⁺/H⁺ exchanger, and depleted <i>Slc9a3</i> in male mice causes infertility due to the abnormal dilated lumen of the rete testis and efferent ductules. Furthermore, SLC9A3 interacts with CFTR in the pancreatic duct and functions as a genetic modifier of CF. However, SLC9A3 function and its relation to CFTR expression in the male reproductive tract in vivo remain elusive. In the present study, we found that CFTR expression was dramatically decreased in the epididymis and vas deferens of <i>Slc9a3</i> knockout mice. Adult <i>Slc9a3</i>^<i>-/-</i> mice showed not only significantly decreased epididymis and vas deferens weight but also increased testis weight. Furthermore, <i>Slc9a3</i>^<i>-/-</i> mice developed obstructive azoospermia because of abnormal abundant secretions and calcification in the lumen of the reproductive tract. Ultrastructural analysis of the epithelium in <i>Slc9a3</i>^{<i>–/–</i>}epididymis and vas deferens displayed disorganized and reduced number of stereocilia and numerous secretory apparatuses. Our data revealed that interdependence between SLC9A3 and CFTR is critical for maintaining a precise microenvironment in the epithelial cytoarchitecture of the male reproductive tract. The <i>Slc9a3</i>-deficient mice with impaired male excurrent ducts in this study provide proof for our clinical findings that some Taiwanese of CBAVD carry <i>SLC9A3</i> deletion but without major <i>CFTR</i> mutations.</p></div

The lumen of the efferent ductules of 4-month-old <i>Slc9a3</i>^<i>-/-</i> males is dilated and calcified.

<p>Comparison of the efferent ducts of 4-month-old WT (A, B) and <i>Slc9a3</i>^<i>-/-</i> (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)</p

Loss of SLC9A3 induces testicular atrophy.

<p>Testicular histology was assessed by H&E staining of testis sections of 6-month-old WT mice (A, B) and <i>Slc9a3</i>^<i>-/-</i> 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) <i>Slc9a3</i>-deficient mice. Vacuolation of seminiferous epithelia was observed in the seminiferous tubules of <i>Slc9a3</i>^<i>-/-</i> 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.</p

Proposed working model of the role of SLC9A3 in the pathogenesis of the male reproductive system.

<p>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 <i>Slc9a3</i>^<i>-/-</i> 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.</p

SLC9A3 expression in the epithelium of male excurrent ducts of 2-month-old WT mice.

<p>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.</p

Absence of spermatozoa in the caput epididymis of adult <i>Slc9a3</i>^<i>-/-</i> mice.

<p>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) <i>Slc9a3</i>^<i>-/-</i> 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 <i>Slc9a3</i>^<i>-/-</i> male mice. Scale bar = 200 μm (A, C, E, G) and 20 μm (B, D, F, H).</p

Comparison of fertile capability between wild-type, heterozygous, and homozygous <i>Slc9a3</i> knockout mice.

<p>Comparison of fertile capability between wild-type, heterozygous, and homozygous <i>Slc9a3</i> knockout mice.</p

CFTR is prominently reduced in the epididymis and vas deferens of <i>Slc9a3</i>^-/- mice.

<p>(A, B, C) CFTR expression in the caput (A) and cauda (B) epididymis and vas deferens (C) of 2-month-old <i>Slc9a3</i>^<i>-/-</i> 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 <i>Slc9a3</i>^<i>-/-</i> 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 <i>Slc9a3</i>^<i>-/-</i> mice. Each bar represents the mean ±SEM; <i>n</i> = 3 per genotype. *Significant difference compared with WT mice (***p < 0.0001, analyzed using the Student’s <i>t</i> test).</p

Aberrant secretions within the lumen of the vas deferens in <i>Slc9a3</i>^<i>-/-</i> males.

<p>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).</p

Gross morphology of the reproductive organs of infertile <i>Slc9a3</i>^<i>-/-</i> male mice.

<p>(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 <i>Slc9a3</i> 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; <i>n</i> = 10 per genotype. *Significant difference compared with WT mice (*p < 0.05 and ***p < 0.0001, analyzed using the Student’s <i>t</i> test).</p