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Doctor of Philosophy (PhD)
Molecular and Cellular Biology
Year Degree Awarded
RAFAEL A. FISSORE
PABLO E. VISCONTI
ALEJANDRO P. HEUCK
Cell Biology | Developmental Biology
Ca2+ signaling induced at fertilization, also known as oscillations, is essential in mammalian eggs to initiate early embryonic development. The generation of the oscillations relies on optimization of Ca2+ toolkit components during oocyte maturation. In this dissertation, we intend to deepen our understanding of how this differentiation of the Ca2+ toolkit, especially those components associated with Ca2+ influx, is achieved during maturation, and how it contributes to the filling of the ER Ca2+ store during the maturation and fertilization.
We first identified the expression and characterized the function of the components of the Store Operated Ca2+ Entry (SOCE) during oocyte maturation. We observed that SOCE underwent suppression during maturation concomitant with an increase in [Ca2+] ERcontent. We demonstrated that the suppression of SOCE coincided with the inability of Stim1, the Ca2+ sensor in ER, to form puncta near the PM, which prevented interaction with Orai1, the channel on the PM. Consistent with a possible role on Ca2+ homeostasis in oocytes, overexpression of Stim1 and Orai1 increased basal Ca2+ levels during maturation, especially during the GV stage, but this influx was suppressed in MII eggs. These results suggest that Ca2+ uptake during maturation is closely related to the Ca2+ content of the ER. Bypassing this inactivation via expression of mutant versions of Stim1 prevented oocytes from resuming meiosis.
The inactivation of SOCE was due in part to changes in Stim1 organization during maturation. We found that Stim1 reorganization was occurred largely due to CDK1. We confirmed the effect of phosphorylation by expressing several non-phosphorylated mutants of Stim1. These mutants displayed cortical location and puncta, and interact with Orai1, and enhanced Ca2+ influx at the MII stage.
Thus, our study also demonstrates that Ca2+ influx and SOCE are actively regulated during mouse oocyte maturation by the MII stage, the stage of fertilization. The suppression of SOCE relies on phosphorylations on the C-terminal end of Stim1 by CDK1.Therefore our studies show that down-regulation of Ca2+ influx is required for oocyte maturation and meiotic progression, although it is still unclear how the sperm manages to re-activate Ca2+ influx after fertilization, and what channel(s) underlie Ca2+ influx during fertilization. Future studies should address how this is accomplished and the channel(s) that mediate it.
Cheon, Banyoon, "CHARACTERIZATION OF Ca2+ INFLUX PATHWAY(S) DURING MOUSE OOCYTE MATURATION" (2014). Doctoral Dissertations. 167.