The purpose of the egg is to give rise to offspring in sexually reproducing organisms. Zinc thresholds became connected to egg quality from initial breakthrough discoveries in M. musculus which demonstrated that large-scale zinc fluxes occur during meiotic maturation, and these fluxes are required to maintain female egg viability. Numerous reproductive processes are conserved in multiple areas of the phylogenetic tree, thus we hypothesized that large-scale zinc fluxes are a conserved characteristic that is required for female egg viability in the invertebrate Caenorhabditis elegans. C.elegans are a powerful model system to conduct zinc studies because they possess a simple reproductive system and provide the ability to easily harvest and visualize developing eggs. Initial experiments demonstrated that hermaphrodites cultured under zinc insufficient conditions produced fewer oocytes which resulted in a reduced brood size. Oocytes maturing in vitro under zinc insufficient conditions were unable to extrude the second polar body at the end of Meiosis II, resulting in aberrant pronuclear formation, hyperploidy, spindle defects and abnormal cytokinesis. These combined defects resulted in cell cycle arrest at various time-points between pronuclear migration and the 2-cell mitotic stage. These findings provided the basis for uncovering the presence of zinc fluxes in maturing C. elegans oocytes. Utilizing the combined approaches of X-ray Fluorescence Microscopy and fluorescence imaging with the novel zinc sensor ZincBy-1, we discovered that C. elegans zygotes exhibit large-scale zinc fluxes during meiotic progression at stages similar to mouse, with influx occurring after fertilization from Metaphase I to Metaphase II. Efflux occurred from Anaphase II through Pronuclear Fusion, after which point zinc levels remained steady through the 2-cell stage. XFM data of total zinc corresponds to the changing levels highlighted by ZincBy-1. Large-scale zinc fluxes were restricted to zygotes during meiotic progression, as we did not detect this activity during oocyte maturation or in the distal, loop or proximal regions of the spatio-temporal gonad. Furthermore, zinc sequestration at the influx stage induces developmental abnormalities including retraction or retention of the second polar body, hyperploidy, and mitotic spindle defects that ultimately lead to cell cycle arrest. When I tracked zinc movement throughout meiotic maturation, I discovered that labile zinc is first present in the cytoplasm during influx through Metaphase II and then continuously exits the cytoplasm into multiple eggshell layers during efflux from Anaphase II through pronuclear fusion. Combined these results support that the hypothesis that dynamic zinc fluxes are conserved between C. elegans and mammals during meiotic maturation. However, key details revealed that zinc regulation in C. elegans diverges from M. musculus. First, in C. elegans, fertilization occurs before zinc influx is initiated, however fertilization does not occur prior to influx in mouse. Second, zinc efflux occurs independently of meiotic arrest in C. elegans, unlike mouse where Metaphase II arrest occurs prior to the zinc spark. Collectively, my studies in C. elegans have demonstrated that zinc availability strongly impacts oogenesis, proper meiotic spindle assembly and cytokinesis. Zinc fluxes are a key aspect for proper meiotic progression. Zinc influx occurs during a critical developmental window during early meiosis, where zinc ion absorption increases by 470%. Without accrual of adequate zinc during this period, the maturing oocyte cannot properly complete meiosis and is inviable.

Last modified

• 02/11/2019

Creator

• Adelita D. Mendoza

DOI

• https://doi.org/10.21985/N2ZR15

Subject

• Interdepartmental Biological Sciences (IBiS) Graduate Program

Keyword

• germline regulation
• zinc
• zinc flux
• oocyte maturation

Date created

• 2017-01-01

Resource type

• Dissertation

Rights statement

• In Copyright