Identification of vesicles, their contents, and how they may contribute to oocyte quality and female fertility
For decades oocyte organelles have undergone considerable investigation as a way to understand their contribution to reproductive competence. However, despite their frequent observation in oocyte studies, little attention has been paid to an understanding of oocyte derived vesicles. It is understood that vesicle volume appears to decline throughout oocyte maturation which suggests the possible use, and importance of its contents during this process. While few have fortuitously identified the contents of oocyte vesicles, the lack of reproducible evidence and assertive conclusions prevents any formal certainty of both their role and what they contain. Based on these limited findings, the aim of the current study was to not only identify oocyte derived vesicles, but to uncover and recognise the composition of said vesicles in a centrifuged oocyte model in order to explore their attributes that may be further utilised to improve oocyte quality and thus, fertility. Sheep oocytes sourced from the abattoir were either in vitro matured or maintained at germinal vesicle stage before centrifugation to segregate organelles by density into lipid, vesicle, mitochondria, and smooth endoplasmic reticulum layers. Transmission electron microscopy of fixed and sectioned oocytes was performed for an analysis of organelle segregation, and to confirm the location of the organelle layers. Potential vesicle contents were investigated via confocal microscopy where the application of LysoTracker Red, Fluo-4-AM and Nile Red stains were used to detect for acidic organelles, calcium stores and lipids respectively. Stains observed through confocal microscopy were correlated to their respective organelle layer established through transmission electron microscopy. Mature oocytes were significantly more able to layer their organelles, following centrifugation, in comparison to their immature counterparts (p<0.0001). Furthermore, the ability of organelles to layer appeared to be unaffected by either ewe age, or whether oocytes were surrounded by a layer of supporting cumulus cells. Fluorescent imaging of oocytes upon the application of LysoTracker Red or Fluo-4-AM appeared to show no evident staining in relation to vesicles, moreover, visibly highlighting lysosomes and mitochondria respectively. Comparably, Nile Red was expectantly observed to emit fluorescence within the lipid droplets themselves, however, a weaker yet notable emission was additionally apparent within that of the vesicle layer. Bright spot analysis identified two dissimilar lipid profiles between these organelles. The findings of the current study bear no evidence that vesicles are acidic storage sites, nor do the results support the potential of oocyte derived vesicles to possess calcium. The ability of Nile Red to identify chromatically distinct lipid profiles between the vesicle and lipid droplet regions suggests that oocyte derived vesicles are likely to harbour an additional lipid source that is utilised during the process of oocyte maturation, and hence may contribute to the overall developmental competence of the oocyte.
Advisor: Reader, Karen
Degree Name: Bachelor of Biomedical Sciences with Honours
Degree Discipline: Pathology
Publisher: University of Otago
Keywords: Oocyte; Oocyte quality; Assisted reproduction; In vitro maturation; Vesicles; Oocyte derived vesicles
Research Type: Thesis