Proper acrosome formation is critical for sperm functionality and morphology, involving dynamic processes such as organelle relocation, repurposing, and reshaping. The cytoskeleton provides essential mechanical support and facilitates intracellular trafficking during this process. Traditional studies utilizing thin histological sections often fail to capture the three-dimensional (3D) context of cells and tissues, potentially leading to information loss.
To address this limitation, we have developed a tissue-clearing protocol based on the CLARITY method, combined with confocal microscopy, to study spermatogenesis in 3D within the seminiferous tubules. Using Imaris software, we reconstruct the surfaces of postmeiotic nuclei and acrosomes at various stages of spermatogenesis (Golgi, Cap, Acrosomal, and Maturation phase) and quantify parameters such as volume, sphericity, and distance from the tubule borders. These quantitative data serve as valuable inputs for computational comparisons and modelling.
Our previous research has identified a link between the nuclear protein Protamine 2 (Prm2) and the cytoskeletal protein Septin 12 (Sanovec et al., 2024). In Prm2-deficient (Prm2−/−) mice, we observed aberrant acrosome formation and altered localization of Septin 12 and actin, suggesting a disruption in cytoskeletal organization during spermiogenesis (Schneider et al., 2016). Therefore, we aim to apply our 3D imaging approach to analyze acrosome formation in Prm2−/− mice in detail, focusing on the spatial distribution of actin and Septin 12 as well as the acrosome as a whole organelle. Our findings will be further supported using super-resolution and electron microscopy.
This integrative approach provides a comprehensive understanding of acrosome biogenesis and its perturbations due to Protamine 2 deficiency, offering insights into the molecular mechanisms underlying sperm development and potential causes of male infertility. The newly developing platform can act as a robust pipeline for acrosome analysis in various mouse models, uncovering subtle details in this dynamic process.