Document Type

Open Access Thesis

Embargo Period

8-30-2017

Degree Program

Molecular & Cellular Biology

Degree Type

Master of Science (M.S.)

Year Degree Awarded

2017

Month Degree Awarded

September

Abstract

In mammals, calcium influx is required for oocyte maturation and egg activation, as it supports the persistent calcium oscillations induced by fertilization. These oscillations are required for the initiation of embryo development. The molecular identities of the plasma membrane calcium-permeant channels that underlie calcium influx are not established. Among these channels, Transient Receptor Potential Vanilloid, member 3 (TRPV3) allows divalent cations, namely strontium (Sr2+) and calcium (Ca2+) with high permeability, into cells, and its expression pattern seems to predict an essential role in the initiation of development. Another channel that was identified to be expressed in oocytes/eggs is the low-voltage-activated T-type channel, CaV3.2. However, the ability to accurately probe the expression and function of these channels on Ca2+ homeostasis in mouse eggs is hindered by the lack of specific and known pharmacological agents and antibodies for these channels.

Here, we simultaneously knockout out these two Ca2+ influx channels in the mouse to explore the effects on Ca2+ homeostasis. We examined fertility rates, development, and morphological defects that arose from the double null pups. Next, we investigated the consequences on Ca2+ store content in immature and mature oocytes and eggs. We also examined the effects on fertilization-induced Ca2+ oscillations in response to in vitro fertilization and PLCz cRNA microinjection. We found that female mice null for these channels display drastic subfertility compared to the single knockout mice for these channels. Additionally, the Ca2+ store content is significantly diminished in double knockout eggs versus controls, as was the frequency of the fertilization-induced Ca2+ oscillations. These results suggest that these channels play a crucial role in Ca2+ influx during maturation and contribute to maintain Ca2+ oscillations post-fertilization. These null oocytes and eggs will be an important tool to perform electrophysiological studies to accurately measure the native current(s) of a specific channel(s) in eggs, and to identify the channel(s) that mediate Ca2+ during fertilization.

First Advisor

Rafael Fissore

Second Advisor

Dominique Alfandari

Third Advisor

Craig Albertson

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