High-Resolution 3D Reconstruction of Human Oocytes Using Focused Ion Beam Scanning Electron Microscopy

Abstract

The egg plays a pivotal role in the reproduction of our species. Nevertheless, its fundamental biology remains elusive. Transmission electron microscopy is traditionally used to inspect the ultrastructure of female gametes. However, two-dimensional micrographs contain only fragmentary information about the spatial organization of the complex oocyte cytoplasm. Here, we employed the Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) to explore human oocyte intracellular morphology in three dimensions (3D). Volume reconstruction of generated image stacks provided an unprecedented view of ooplasmic architecture. Organelle distribution patterns observed in nine donor oocytes, representing three maturational stages, documented structural changes underlying the process by which the egg acquires developmental competence. 3D image segmentation was performed to extract information about distinct organelle populations, and the following quantitative analysis revealed that the mitochondrion occupies ∼ 4.26% of the maturing oocyte cytoplasm. In summary, this proof-of-concept study demonstrates the potential of large volume electron microscopy to study rare samples of delicate female gametes and paves the way for applying the FIB-SEM technique in human oocyte research.

The ultrastructural nature of human oocytes’ cytoplasmatic abnormalities and the role of cytoskeleton dysfunction

“…However, no studies tackled to address molecular mechanisms underlying the emergence of different cytoplasmic irregularities in developing human oocytes. We have previously mapped out ultrastructural changes during human oocyte maturation, and our 2D and 3D image data documented major reorganization of ooplasm in preparation for fertilization (23,24). Correct positioning and active movement of organelles within the cytoplasm rely on their interaction with cytoskeleton components.…”

“…High magnification is needed to detect pathological features such as misshaped mitochondria and enlarged SER. The development of high-resolution tomographic methods now enables volumetric imaging of oocyte and ovarian tissue architecture (24,53). Three-dimensional reconstruction of isolated and/or cumulusenclosed dysmorphic oocytes would elucidate the spatial relationship of subcellular structures and advance our understanding of how the oocytes interact with surrounding cells.…”

“…In this study, we harnessed our experience with electron microscopy of normal human oocytes (23,24) and examined the subcellular morphology of dysmorphic female gametes rejected for ICSI. To elucidate cellular mechanisms implicated in the evolution of cellular aberrations, we experimentally perturbed cytoskeleton function in oocytes maturing in vitro and inspected their inner morphology.…”

The 3D Controllable Fabrication of Nanomaterials with FIB-SEM Synchronization Technology

“…Heymann et al [ 133 ] first reported the 3D volume reconstruction of biological specimens for yeast cells. Trebichalská et al [ 134 ] reconstructed an unprecedented view of ooplasmic architecture and observed organelle distribution patterns in nine donor oocytes’ developmental competence. 3D image segmentation was performed to extract information.…”