Reproductive pathology and sperm physiology in acid sphingomyelinase-deficient mice

Abstract

Types A and B Niemann-Pick disease (NPD) are lysosomal storage disorders resulting from the deficient activity of acid sphingomyelinase (ASM). In this manuscript we report the pathobiology of male gonadal tissue and sperm in a knockout mouse model of NPD and demonstrate the importance of ASM for normal sperm maturation and function. Characteristic lipid-filled vacuoles were evident in light micrographs of testis' seminiferous tubules and epithelial cells lining the epididymis of -/- mice. Electron micrographs extended these findings and revealed storage vesicles within Sertoli cells of the seminiferous tubules. Mature spermatozoa from -/- mice showed marked ASM deficiency and elevated levels of sphingomyelin and cholesterol. Flow cytometric analysis revealed that affected spermatozoa had disrupted plasma and acrosome membranes, and mitochondrial membrane depolarization. They also did not undergo proper capacitation. Morphological abnormalities such as kinks and bends at the midpiece-principle piece junction were evident in spermatozoa from affected mice, with consequent deficits in motility. Notably, the mutant sperm regained normal morphology on incubation in mild detergent, demonstrating that the bending defects were a direct consequence of membrane lipid accumulation. A mechanism for these abnormalities is proposed that suggests lipid accumulation in the gonads results in regulatory volume decrease defects within the developing sperm, and that regulatory volume decrease defects, in turn, lead to the observed abnormalities in sperm morphology and function. These results provide in vivo evidence that ASM activity plays a critical role in sperm maturation and function, and a basis for similar studies in sexually mature, male NPD patients.

Figure 1.

Lipid accumulation in gonads at 6 months of age. A: H&E-stained sections of testis (original magnification, ×1000) and epididymis (original magnification, × 200). Note the areas of lipid accumulation (arrows) in Sertoli cells (Se), interstitial (IS), epididymal lumen (Lu), epididymal intertubular space (IT), and stroma (St). G, germ cells; Sp, spermatozoa. B: Electron micrographs of testis and epididymides. Note multilamellar vesicles (arrows) in all cell types. Sm, smooth muscle cells; B, basal cells; P, principle cells; Lu, lumen. Original magnifications: × 2000.

Figure 1.

Lipid accumulation in gonads at 6 months of age. A: H&E-stained sections of testis (original magnification, ×1000) and epididymis (original magnification, × 200). Note the areas of lipid accumulation (arrows) in Sertoli cells (Se), interstitial (IS), epididymal lumen (Lu), epididymal intertubular space (IT), and stroma (St). G, germ cells; Sp, spermatozoa. B: Electron micrographs of testis and epididymides. Note multilamellar vesicles (arrows) in all cell types. Sm, smooth muscle cells; B, basal cells; P, principle cells; Lu, lumen. Original magnifications: × 2000.

Figure 2.

Biochemical abnormalities of caudal spermatozoa at 6 months of age. A: SPM accumulation in whole cell lysates. B: HPLC quantitation of ASM activity in whole cell lysates. C: Thin layer chromatogram showing ASM activity in homogenates, detergent soluble supernatants, and detergent insoluble pellets. D: Sphingomyelinase activity determined in situ and analyzed by flow cytometry. “Chase” indicates the extent of exogenous SPM degradation after initial labeling of cells (“Pulse”). E, Sphingomyelinase activity determined in situ and assessed by thin layer chromatography. Chase indicates the extent of exogenous SPM degradation after initial labeling of cells (Pulse). Ceramide is a metabolic product of SPM.

Figure 3.

Cholesterol accumulation in caudal spermatozoa at 6 months of age. A: Representative freeze-fracture PF-face images of filipin-sterol complexes, FSC (original magnifications, ×10,000; insets: ×60,000). B: Surface analysis of FSC. The steepness of the spectral slope is indicative of image surface coarseness. C: In situ plasma membrane cholesterol efflux.

Figure 4.

Morphological abnormalities of caudal spermatozoa at 6 months of age. Spermatozoon tail retroflexion: <90° (arrow 1), ∼90° (arrow 2), and 180° (arrow 3). Original magnifications: × 400.

Figure 5.

Physiological abnormalities of caudal spermatozoa at 6 months of age by flow cytometric analysis. A: Plasma membrane integrity. The percentage of healthy, plasma membrane intact spermatozoa are shown in the lower right quadrants of the density dot-blot profiles. B: Acrosome status. The percentage of acrosome-intact spermatozoa is shown in the lower right quadrants of the density dot-blot profiles. C: Mitochondrial membrane potential. The percentage of metabolically active sperm is shown in the upper right quadrants of the density dot-blot profiles. D: NO production. The percentage of spermatozoa producing NO as a function of plasma membrane integrity is shown in the upper right quadrants of the density dot-blot profiles. E: Phosphatidylserine bilayer translocation. The percentage of spermatozoa with intact plasma membranes undergoing PS translocation from the inner to the outer leaflet of the plasma membrane is indicated in the lower left quadrants of the density dot-blot profiles.