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.

Department of Histology and Embryology Faculty of Medicine Masaryk University Brno Czechia

Reprofit International Brno Czechia

TESCAN ORSAY HOLDING a s Brno Czechia

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Bianchi S., Macchiarelli G., Micara G., Linari A., Boninsegna C., Aragona C., et al. (2015). Ultrastructural markers of quality are impaired in human metaphase II aged oocytes: a comparison between reproductive and in vitro aging. J. Assist. Reprod. Genet. 32 1343–1358. 10.1007/s10815-015-0552-9 PubMed DOI PMC

Coelho S., Pires-Luís A. S., Oliveira E., Alves Â, Leal C., Cunha M., et al. (2020). Stereological study of organelle distribution in human oocytes at metaphase I. Zygote 28 308–317. 10.1017/S0967199420000131 PubMed DOI

Darbandi S., Darbandi M., Khorram Khorshid H. R., Sadeghi M. R., Agarwal A., Sengupta P., et al. (2017). Ooplasmic transfer in human oocytes: efficacy and concerns in assisted reproduction. Reprod. Biol. Endocrinol. 15:77. 10.1186/s12958-017-0292-z PubMed DOI PMC

Holubcová Z., Kyjovská D., Martonová M., Páralová D., Klenková T., Kloudová S. (2019). Human egg maturity assessment and its clinical application. J. Vis. Exp. 150:e60058. 10.3791/60058 PubMed DOI

Hrnčíř T., Lopour F., Zadražil M., Delobbe A., Salord O., Sudraud P. (2012). “Novel plasma FIB/SEM for high speed failure analysis and real time imaging of large volume removal,” in Proceedings of the ISTFA 2012: 38th International Symposium for Testing and Failure Analysis, (Phoenix: ASM International; ), 620.

Hrnčíř T., Šikula M., Oboňa J. V., Gounet P. (2016). “How to Achieve Artifact-Free FIB Milling on Polyimide Packages,” in Proceedings of the ISTFA 2016: 42nd International Symposium for Testing and Failure Analysis, (Fort Worth, TX: ASM International; ), 630–634.

Kizilyaprak C., Stierhof Y. D., Humbel B. M. (2019). Volume microscopy in biology: FIB-SEM tomography. Tissue Cell 57 123–128. 10.1016/j.tice.2018.09.006 PubMed DOI

Mtango N. R., Potireddy S., Latham K. E. (2008). Oocyte quality and maternal control of development. Int. Rev. Cell Mol. Biol. 268 223–290. 10.1016/S1937-6448(08)00807-1 PubMed DOI

Otsuki J., Nagai Y., Chiba K. (2007). Lipofuscin bodies in human oocytes as an indicator of oocyte quality. J. Assist. Reprod. Genet. 24 263–270. 10.1007/s10815-007-9130-0 PubMed DOI PMC

Pires-Luís A. S., Rocha E., Bartosch C., Oliveira E., Silva J., Barros A., et al. (2016). A stereological study on organelle distribution in human oocytes at prophase I. Zygote 24 346–354. 10.1017/S0967199415000258 PubMed DOI

Sá R., Cunha M., Silva J., Luís A., Oliveira C., Teixeira da Silva J., et al. (2011). Ultrastructure of tubular smooth endoplasmic reticulum aggregates in human metaphase II oocytes and clinical implications. Fertil. Steril. 96 143.e7–149.e7. 10.1016/j.fertnstert.2011.04.088 PubMed DOI

Sathananthan A. H. (1994). Ultrastructural changes during meiotic maturation in mammalian oocytes: unique aspects of the human oocyte. Microsc. Res. Tech. 27 145–164. 10.1002/jemt.1070270208 PubMed DOI

Schindelin J., Arganda-Carreras I., Frise E., Kaynig V., Longair M., Pietzsch T., et al. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9 676–682. 10.1038/nmeth.2019 PubMed DOI PMC

Sundström P., Nilsson B. O., Liedholm P., Larsson E. (1985). Ultrastructure of maturing human oocytes. Ann. N.Y. Acad. Sci. 442 324–331. 10.1111/j.1749-6632.1985.tb37536.x PubMed DOI

Trebichalská Z., Kyjovská D., Kloudová S., Otevřel P., Hampl A., Holubcová Z. (2021). Cytoplasmic maturation in human oocytes: an ultrastructural study †. Biol. Reprod. 104 106–116. 10.1093/biolre/ioaa174 PubMed DOI PMC

Watson A. J. (2007). Oocyte cytoplasmic maturation: a key mediator of oocyte and embryo developmental competence. J. Anim. Sci. 85(13 Suppl) E1–E3. 10.2527/jas.2006-432 PubMed DOI

Xu C. S., Hayworth K. J., Lu Z., Grob P., Hassan A. M., García-Cerdán J. G., et al. (2017). Enhanced FIB-SEM systems for large-volume 3D imaging. Elife 6:e25916 10.7554/eLife.25916 PubMed DOI PMC

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