The Role of Chorein Deficiency in Late Spermatogenesis

Abstract

VPS13A, also known as chorein, whose loss of function causes chorea-acanthocytosis (ChAc), is characterized by Huntington's-disease-like neurodegeneration and neuropsychiatric symptoms in addition to acanthocytosis in red blood cells. We previously reported that ChAc-model mice with a loss of chorein function exhibited male infertility, with asthenozoospermia and mitochondrial dysmorphology in the spermatozoa. Here, we report a novel aspect of chorein dysfunction in male fertility, particularly its role in spermatogenesis and mitochondrial integrity. An increase in anti-malondialdehyde antibody immunoreaction within the testes, predominantly observed at the advanced stages of sperm formation in chorein-deficient mice, suggests oxidative stress as a contributing factor to mitochondrial dysfunction and impaired sperm maturation. The chorein immunoreactivity in spermatids of wild-type mice accentuates its significance in sperm development. ChAc-model mice exhibit mitochondrial ultrastructural abnormalities, specifically during the late stages of sperm maturation, suggesting a critical timeframe for chorein's action in spermiogenesis. We observed an increase in TOM20 protein levels, indicative of disrupted mitochondrial import mechanisms. The concurrent decrease in metabolic enzymes such as IDH3A, LDHC, PGK2, and ACAT1 suggests a complex chorein-mediated metabolic network that is essential for sperm vitality. Additionally, heightened separation of cytoplasmic droplets from sperm highlights the potential membrane instability in chorein-deficient spermatozoa. Metabolomic profiling further suggests a compensatory metabolic shift, with elevated glycolytic and TCA-cycle substrates. Our findings suggest that chorein is involved in anti-ferroptosis and the maturation of mitochondrial morphology in the late stages of spermatogenesis, and its deficiency leads to asthenozoospermia characterized by membrane instability, abnormal cytosolic glycolysis, abnormal mitochondrial function, and a disrupted TCA cycle. Further analyses are required to unravel the molecular mechanisms that directly link these findings and to elucidate the role of chorein in spermatogenesis as well as its broader implications.

Keywords: IDH3A; TOM20; VPS13A; asthenozoospermia; chorea-acanthocytosis; chorein; ferroptosis; mitochondria.

Figure 1

Transmission electron microscopy of mitochondria during spermatogenesis in wild-type (WT) and ChAc^Del/Del mice. Left column: Early-stage spermatids in the seminiferous tubule, with no discernible differences between the mitochondria of WT and ChAc^Del/Del mice. Middle column: Late-stage spermatids in the seminiferous tubule, where the mitochondria of ChAc^Del/Del mice exhibit distorted cross-sections. Right column: Epididymal spermatozoa, showing morphological distortions and changes in electron density within the mitochondria of ChAc^Del/Del mice. Scale bars = 1 µm.

Figure 2

Results of immunoprecipitation analysis. Seven proteins were identified as candidate chorein-interacting proteins by proteomic analysis based on the spectral counting method. IDH3A and ACAT1 were detected by Western blotting analysis following chorein co-IP. Chorein and PGK2 were detected by Western blotting analysis following IDH3A co-IP.

Figure 3

Western blotting analysis for epididymal sperm and testis lysates was performed to compare the protein levels of ChAc^Del/Del mice (n = 5) with those of WT mice (n = 5). (A) Proteins were visualized using the MemCode Reversible Protein Stain Kit after separation by SDS-PAGE and transfer to PVDF membranes. The contrast of the sperm lysates image was increased by a factor of two to enhance visibility. Sperm lysates exhibited different patterns of protein expression between WT and ChAc^Del/Del mice. Testis lysates exhibited similar patterns between both groups. (B) Representative results of Western blotting analysis are shown. (C) Relative quantification of Western blotting analysis is presented with white and black bars indicating WT and ChAc mice, respectively. The Mann–Whitney U test was performed for statistical analysis. WT sperm lysates exhibited significantly higher levels of IDH3A and PGK2 than ChAc^Del/Del sperm lysates. Interestingly, WT mice exhibited significantly lower levels of TOM20 than ChAc^Del/Del mice. No differences were observed between WT and ChAc^Del/Del testis lysates. * p < 0.05; ^† p < 0.01.

Figure 4

Metabolites of the glycolysis and TCA-cycle pathways obtained from metabolomic analysis. Bar graphs represent the relative amounts of metabolites. White and black bars indicate WT mice (n = 5) and ChAc mice (n = 5), respectively. White boxes represent metabolites. Black boxes represent enzymes. PGK2, LDHC, and IDH3A are enzymes detected by chorein co-IP and proteomic analysis of sperm lysates; Welch’s t-test was performed. * p < 0.05; ^† p < 0.01; N.D., not detected. Abbreviations; 1,3DPG, 1,3-bisphosphoglycerate; 2OG, 2-oxoglutarate; 2PG, 2-phosphoglyceric acid; 3PG, 3-phosphoglyceric acid; G3P, glyceraldehyde 3-phosphate; G6P, glucose-6-phosphate; GAP, glyceraldehyde 3-phosphote; F1,6P, fructose 1,6-bisphosphate; F6P, fructose 6-phosphate; PEP, phosphoenolpyruvate.

Figure 5

(A) Localization of chorein, PGK2, LDHC, and IDH3A proteins in the seminiferous tubule/ductus epididymis. Small windows show magnified images. Chorein, PGK2, and LDHC were each stained with appropriate antibodies (brown). The nuclei were stained with hematoxylin (blue). Cytoplasm in elongated spermatids and cytoplasmic droplets in epididymal sperm were stained with antibodies to the PGK2 protein. The pattern of LDHC protein immunoreaction was consistent with that of PGK2. Immunolabeled IDH3A protein (red) was observed in spermatogonia but not in spermatids or epididymal sperm. The acrosome sacs and nuclei were stained with PNA (green) and DAPI (blue), respectively. Scale bars = 50 µm. (B) Localization of chorein and IDH3A in epididymal sperm. Chorein (green) was observed in the midpiece, and IDH3A (red) was observed at both ends of the midpiece. The nuclei were stained with DAPI (blue). Scale bars = 10 µm. (C) The majority of the sperm of ChAc mice showed no immunoreactivity of the IDH3A protein. Scale bars = 10 µm.

Figure 6

Immunohistochemical assay of malondialdehyde (MDA) in the seminiferous tubules. MDA immunoreactions (green) were observed predominantly in the final stages of spermatogenesis in both wild-type (WT) and ChAc^Del/Del mice. The magnified image of the region enclosed by the dashed lines is shown on the left side. In WT mice, the cilia were primarily lightly stained, whereas in ChAc^Del/Del mice, there was relatively strong staining in the region encompassing the midpiece of the sperm. The nuclei were stained with DAPI (blue). Scale bars = 20 µm.