Three-dimensional reconstruction of rat sperm using volume electron microscopy

Abstract

Three-dimensional (3D) reconstruction serves as a crucial instrument for the analysis of biological structures. In particular, a comprehensive and accurate 3D ultrastructural examination of rat sperm is vital for understanding and diagnosing male fertility issues and the underlying causes of infertility. In this study, we utilize the automated tape-collecting ultramicrotome scanning electron microscopy (ATUM-SEM) imaging technique, which is a highly effective method for 3D cellular ultrastructural analysis. Our findings reveal that during spermiogenesis, the volume of the nucleus significantly decreases, shrinking to just 10% of its original size. The acrosomal vesicles derived from the Golgi apparatus converge and elongate along the spermatid nucleus. These vesicles then attach to the nucleus via a cap-like structure, thereby defining the head side of the spermatozoa. In the initial stages of spermiogenesis, the mitochondria in spermatids are distributed beneath the cell membrane. As the process progresses, these mitochondria gradually migrate to the sperm tail, where they form the mitochondrial sheath. This sheath plays a crucial role in providing the energy required for the movement of the sperm. In addition, we reconstruct the mRNA-stroring structure-chromatoid body in sperm cells, which are cloud-like or net-like structures in the cytoplasm. The precise and comprehensive nature of 3D ultrastructural examination allows for a deeper understanding of the morphological process of spermiogenesis, thereby contributing to our knowledge of male fertility and the causes of infertility. Our research has significantly advanced the understanding of the 3D ultrastructure of sperm more comprehensively than ever before.

1. Introduction

Spermiogenesis is the final stage of spermatogenesis, during which haploid, round spermatids undergo a series of morphological and molecular transformations to become mature spermatozoa. This process involves significant changes in cell morphology, including the formation of the sperm head, neck, and tail structures, and is accompanied by profound alterations in the chromatin structure within the nucleus [1–3]. The acrosome, a specialized organelle that covers the anterior part of the sperm head, is formed from Golgi-derived vesicles that coalesce and elongate along the anterior end of the nucleus. The mature acrosome is a cap-like structure that covers approximately two-thirds of the nucleus surface and is essential for the ability of sperm to penetrate the egg’s outer layers during fertilization [4,5]. The mitochondria in spermatids initially localize beneath the cell membrane, and as spermiogenesis progresses, they migrate toward the developing sperm tail. These mitochondria undergo changes in volume and structure, becoming smaller, and eventually wrap around the central axoneme to form a helical mitochondrial sheath. This sheath is critical for providing the energy required for sperm motility [4,6,7]. Additionally, the centrioles in spermatids migrate and contribute to the formation of the central axoneme of the sperm tail, which is the core structural component of the axoneme. After the complex morphological changes that occur during spermiogenesis, the newly formed spermatozoa are released into the lumen of the seminiferous tubules. They are then transported to the epididymis, where they undergo further maturation processes that are essential for acquiring motility and fertilization capability [8,9].

Our current understanding of spermiogenesis has been largely informed by traditional light microscopy and conventional electron microscopy (EM) techniques [4,9]. These methods, however, have inherent limitations, such as the lack of true three-dimensional (3D) ultrastructural detail, which fails to accurately reflect the complete process of spermiogenesis. Recent advancements in EM-based techniques for 3D ultrastructural reconstruction, particularly in the nervous system, have led to the development of a new category of EM known as volume EM. Volume EM encompasses serial-section electron microscopy (ssET) [10], serial block face scanning electron microscopy (SBEM) [11], focused ion beam scanning electron microscopy (FIB-SEM) [12], automated tape-collecting ultramicrotome scanning electron microscopy (ATUM-SEM) and automated serial-section collection with high-throughput transmission EM, such as GridTape [13,14].