Zinc, an essential trace element that serves as an enzyme cofactor and can also be stored as a divalent ion in cellular vesicles, is emerging as an important mediator of signaling pathways required for biological functions such as growth, development, and reproduction. Zinc mediates cell signaling by acting both as a diffusible ionic signal, similar to calcium ions, and as a covalent modification of selected proteins, similar to phosphorylation. Previous studies from our group have shown that a zinc flux, the movement of labile, biologically available zinc ions across a cell membrane, is required for terminal oocyte maturation, egg-to-zygote transition, and embryonic mitotic divisions. However, roles for zinc fluxes in preceding stages of early ovarian follicle development, when cooperative interactions exist between the oocyte and somatic cells, are unknown. To test the hypothesis that zinc regulates the development of early follicles, we first developed a reliable method for collecting early follicles and their associated germ cells and somatic cells. We then applied single-cell X-ray fluorescence microscopy (XFM), a radioactive zinc tracer, and a labile zinc probe to measure zinc in individual cells within early follicles.Using XFM and a radioactive zinc tracer, we found a significant stage-specific increase and compartmental redistribution of oocyte zinc content upon the initiation of early follicle growth. We discovered active zinc accrual begins early during follicle development. While total zinc content in individual somatic cells is maintained at a constant level, zinc in the oocyte increases in a stage-specific manner: by the secondary follicle stage, the oocyte has nearly a magnitude higher zinc concentration than at the primordial stage. Upon staining whole follicles for labile intracellular zinc, we observed that zinc is located in different subcellular domains in different follicle classes. Specifically, primordial oocytes exhibited localized foci of zinc staining that are colocalized with the ER and Golgi. Oocytes of growing follicles showed a diffuse staining pattern with reduced colocalization with organelles. These zinc fluxes correlated with increased expression of specific zinc transporters, including two that are essential for ooctye maturation, ZIP6 and ZIP10. We further identified clear nuclear and nucleolar expression patterns of the zinc transporters ZnT3 and ZnT5 in the oocyte, suggesting potential zinc mobilization and regulation in the oocyte nucleus. To determine whether zinc plays an instructive role in folliculogenesis, we performed zinc chelation as well as exogenous zinc treatment of neonatal mouse ovaries using an ex vivo culture system. We demonstrated that while oocytes in follicles exhibit high tolerance to pronounced changes in zinc availability, zinc chelation resulted in somatic cell apoptosis, which in turn decreased secondary follicle numbers. On the other hand, exogenous zinc treatment activated the AKT signaling pathway and cell proliferation in the somatic cells of growing follicles, as determined by Ki-67 labeling. Finally, transcriptomic, proteomic, and zinc loading analyses revealed a dynamic regulation of zinc target genes during early follicle development, and an enrichment of zinc targets within the ubiquitination pathway, including the deubiquitinating enzyme UBP5. Taken together, this study developed the first protocol for murine follicle-staged, single-cell isolation that enabled the definition of major zinc physiological dynamics, as well as transcriptomic changes, during early follicle transitions. We demonstrated that a distinct cell type-specific regulation of zinc is required for early gamete development.

Creator

• Chen, Yu-Ying

DOI

• https://doi.org/10.21985/n2-f9ax-0912

Subject

• Developmental biology

Language

• en

Alternate Identifier

• etdadmin_upload_879371
• http://dissertations.umi.com/northwestern:15933

Date created

• 2022-01-01

Resource type

• Dissertation

Rights statement

• In Copyright