Cytoplasmic maturation in human oocytes: an ultrastructural study †

Abstract

Female fertility relies on successful egg development. Besides chromosome segregation, complex structural and biochemical changes in the cytoplasmic compartment are necessary to confer the female gamete the capacity to undergo normal fertilization and sustain embryonic development. Despite the profound impact on egg quality, morphological bases of cytoplasmic maturation remain largely unknown. Here, we report our findings from the ultrastructural analysis of 69 unfertilized human oocytes from 34 young and healthy egg donors. By comparison of samples fixed at three consecutive developmental stages, we explored how ooplasmic architecture changes during meiotic maturation in vitro. The morphometric image analysis supported observation that the major reorganization of cytoplasm occurs before polar body extrusion. The organelles initially concentrated around prophase nucleus were repositioned towards the periphery and evenly distributed throughout the ooplasm. As maturation progressed, distinct secretory apparatus appeared to transform into cortical granules that clustered underneath the oocyteʾs surface. The most prominent feature was the gradual formation of heterologous complexes composed of variable elements of endoplasmic reticulum and multiple mitochondria with primitive morphology. Based on the generated image dataset, we proposed a morphological map of cytoplasmic maturation, which may serve as a reference for future comparative studies. In conclusion, this work improves our understanding of human oocyte morphology, cytoplasmic maturation, and intracellular factors defining human egg quality. Although this analysis involved spare oocytes completing development in vitro, it provides essential insight into the enigmatic process by which human egg progenitors prepare for fertilization.

High-resolution 3D reconstruction of human oocytes using FIB-SEM

“…The 3D reconstruction of the grayscale image stacks (120-1294 slices) provided a new perspective on female gametes' morphology (Figure 1D-F; Video 1,2). In line with previous TEM studies [2,6], the most prominent ultrastructural features we detected involved clusters of small rounded mitochondria, aggregates of the endoplasmic reticulum (ER), and cortical granules (Figure 1D,E). In MI and MII oocytes, chromosomes and microtubule bundles could be recognized in the meiotic spindle area enclosed by organellerich cytoplasm (Figure 1D-F; Videos 1,2).…”

“…The volumetric image data presented here complemented our findings from the previous ultrastructural study [6]. Using the same fixation and sample processing protocol, we could directly compare attributes of our TEM micrographs and FIB-SEM images.…”

“…Ovarian stimulation and oocyte collection were carried out as previously described [6]. Immature oocytes were incubated in vitro until they reached the defined developmental stage.…”

“…Interestingly, all but one sample exhibited prominent electron-dense specs with a diameter reaching up to 7 μm (Figure 2C,F,I; Video 1). These bodies, known as cytoplasmic inclusions, were previously identified as tertiary lysosomes containing degraded intracellular material [6]. Although our FIB-SEM images did not achieve the level of detail seen in TEM micrographs, their 3D reconstruction provided an opportunity to analyze oocyte organelles' spatial interaction (Video 1).…”

“…Results A total of 9 human oocytes (3 germinal vesicle (GV), 3 metaphase I (MI), and 3 metaphase (MII) oocytes) were used to optimize conditions for large volume electron microscopy. The samples were processed using routine TEM protocol [6]. Instead of manual ultramicrotome sectioning, whole oocyte-containing resin blocks were individually inserted into the microscope chamber and subjected to FIB-SEM imaging (Figure 1A).…”