Meiosis is a highly regulated process necessary for proper chromosome division. Zincfluxes regulate mammalian meiosis; between prophase I and metaphase II, total intracellular zinc increases by 50%, while 20% of zinc is released in “zinc sparks” following fertilization. Although zinc fluxes had been shown to be conserved in mammals, it was unknown if they were conserved in different classes of animals. Xenopus laevis, the African clawed frog, was therefore used for these experiments. As a model organism, Xenopus provides a different toolkit than mammals, namely the large size and easy acquisition of its eggs. Both the zinc spark and a decrease in accessible zinc leading to entry into anaphase II were found to be conserved in Xenopus. Additionally, the majority of intracellular manganese is released following fertilization. Most manganese in both eggs and embryos is bound to a low-molecular weight carboxylate. Zinc and manganese are stored in cortical vesicles along with calcium and multiple other transition metals. Both extracellular zinc and manganese act as blocks to polyspermy, though the IC50 of zinc is an order of magnitude less than that of manganese. Because amphibians were the first tetrapod class to evolutionarily diverge, meiotic zinc fluxes are therefore an ancient phenomenon. The manganese release is an unexpected finding, demonstrating a further novel role of a transition metal ion. Compared to mammalian zygotes, proportionally less zinc is released from Xenopus, which is probably due to significantly different egg geometries as well as the large amount of zinc storage in the Xenopus yolk. Multiple transition metals are not stored in the cortical granules of other species’ eggs – it is possible that Xenopus uses them as a system of detoxification. In the second part of this thesis, the zinc binding of EMI2, a meiotic spindle checkpoint protein, was studied. The molecular mechanism by which zinc fluxes regulate meiosis is unknown. EMI2 inhibits the APC/C, an E3 ubiquitin ligase that regulates the cell cycle. EMI2 starts to be expressed when egg zinc levels rise and is degraded following the zinc spark. The protein contains two zinc-binding sites and mutating its zinc-binding region leads to meiotic catastrophe, so it is hypothesized that it is regulated by differential zinc binding. Competitive chelation results show a 420,000-fold difference in binding affinity between EMI2’s two zinc-binding sites. Using highenergy- resolution fluorescence detection spectroscopy, the first binding site (Cys4) binds zinc tighter than the second binding site (Cys3His). The difference in zinc-binding affinities in EMI2 are significantly greater than those found in other RING domain proteins. Although in vitro and in vivo experiments are necessary to determine functionality, these initial results support the hypothesis that EMI2 acts as a zinc-dependent meiotic regulator. The localization and concentration of transition metals in biological samples are mapped using microscopic methods; however, the fixation process can lead to bulk elemental changes and/or alterations in their localization. In the third part of this thesis, the effects of a modified version of Timm’s sulfide staining method were studied to determine if sulfide fixation maintains transition metal content and localization as well as to determine which step(s) of the fixation to resin-infusion process lead to the greatest amounts of elemental change. Chemically-fixed biological samples used for elemental mapping experiments tend to show a significant loss in transition metal content. Aided by the large size of Xenopus eggs, bulk elemental content was analyzed at each step of the fixation and resin-infusion process to determine if and when metal content changed. Surprisingly, sulfide was not necessary for the preservation of either bulk transition metal content or localization in Xenopus eggs, probably because the majority of metals are tightly bound in organelles. Additionally, inductively coupled plasma mass spectrometric analysis showed that microscopy-grade chemicals have enough metal content to contaminate biological samples, demonstrating that sample preparation techniques need to be optimized before starting elemental mapping experiments. Altogether, these studies have revealed new insights into the roles of transition metals in meiosis. Meiotic zinc fluxes are conserved in Xenopus, suggesting that this is an ancient phenomenon. Additionally, a novel manganese release leading to a block to polyspermy was discovered, revealing that other transition metals have important roles in fertilization. Linking intracellular zinc fluxes to possible regulatory pathways, EMI2’s two zinc-binding sites demonstrate the greatest difference in zinc-binding affinities of all previously-studied RING domain proteins, suggesting that one site is regulatory and the other is structural. These results contribute to the paradigm shift that transition metals, rather than only alkali and alkaline earth metals, can serve as signaling agents. Finally, studying elemental changes following chemical fixation in Xenopus eggs demonstrates that tissue type, particularly influenced by the intracellular metal binding environment, strongly affects metal retention. Rarely considered previously, residual metal content in fixation solutions can be enough to lead to significant sample contamination, demonstrating that researchers should analyze the chemicals they use before performing elemental mapping experiments. Six video files are part of this thesis. Their corresponding captions can be found in Appendix A, page 191.

Creator

• Seeler, John Frederick

DOI

• https://doi.org/10.21985/n2-2cre-ta82

Subject

• Inorganic chemistry
• Biology
• Biochemistry

Language

• en

Alternate Identifier

• etdadmin_upload_819316
• http://dissertations.umi.com/northwestern:15573

Keyword

• Xenopus
• Sample Fixation
• Zinc
• Manganese
• Fertilization
• EMI2

Date created

• 2021-01-01

Resource type

• Dissertation

Rights statement

• In Copyright