The ultrastructural nature of human oocytes' cytoplasmic abnormalities and the role of cytoskeleton dysfunction.

OBJECTIVE: To investigate the structural bases of human oocytes' cytoplasmic abnormalities and the causative mechanism of their emergence. Knowledge of an abnormal oocyte's intracellular organization is vital to establishing reliable criteria for clinical evaluation of oocyte morphology. DESIGN: Laboratory-based study on experimental material provided by a private assisted reproduction clinic. SETTING: University laboratory and imaging center. PATIENTS: A total of 105 women undergoing hormonal stimulation for in vitro fertilization (IVF) donated their spare oocytes for this study. INTERVENTIONS: Transmission electron microscopy (TEM) was used to analyze the fine morphology of 22 dysmorphic IVF oocytes exhibiting different types of cytoplasmic irregularities, namely, refractile bodies; centrally located cytoplasmic granularity (CLCG); smooth endoplasmic reticulum (SER) disc; and vacuoles. A total of 133 immature oocytes were exposed to cytoskeleton-targeting compounds or matured in control conditions, and their morphology was examined using fluorescent and electron microscopy. MAIN OUTCOME MEASURES: The ultrastructural morphology of dysmorphic oocytes was analyzed. Drug-treated oocytes had their maturation efficiency, chromosome-microtubule configurations, and fine intracellular morphology examined. RESULTS: TEM revealed ultrastructural characteristics of common oocyte aberrations and indicated that excessive organelle clustering was the underlying cause of 2 of the studied morphotypes. Inhibition experiments showed that disruption of actin, not microtubules, allows for inordinate aggregation of subcellular structures, resembling the ultrastructural pattern seen in morphologically abnormal oocytes retrieved in IVF cycles. These results imply that actin serves as a regulator of organelle distribution during human oocyte maturation. CONCLUSION: The ultrastructural analogy between dysmorphic oocytes and oocytes, in which actin network integrity was perturbed, suggests that dysfunction of the actin cytoskeleton might be implicated in generating common cytoplasmic aberrations. Knowledge of human oocytes' inner workings and the origin of morphological abnormalities is a step forward to a more objective oocyte quality assessment in IVF practice.

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

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.

Perfect date-the review of current research into molecular bases of mammalian fertilization.

Fertilization is a multistep process during which two terminally differentiated haploid cells, an egg and a sperm, combine to produce a totipotent diploid zygote. In the early 1950s, it became possible to fertilize mammalian eggs in vitro and study the sequence of cellular and molecular events leading to embryo development. Despite all the achievements of assisted reproduction in the last four decades, remarkably little is known about the molecular aspects of human conception. Current fertility research in animal models is casting more light on the complexity of the process all our lives start with. This review article provides an update on the investigation of mammalian fertilization and highlights the practical implications of scientific discoveries in the context of human reproduction and reproductive medicine.