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Author ORCID Identifier

https://orcid.org/0000-0002-2599-5361

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Animal Biotechnology & Biomedical Sciences

Year Degree Awarded

2020

Month Degree Awarded

May

First Advisor

Rafael A. Fissore

Second Advisor

Margaret M. Stratton

Third Advisor

Joseph D. Jerry

Fourth Advisor

Pablo E. Visconti

Subject Categories

Cell Biology | Developmental Biology

Abstract

In mammals, fully grown immature oocytes, also known as germinal vesicle (GV), are arrested at prophase of meiosis I. Upon increase of the luteinizing hormone (LH), GV oocytes resume meiosis and reach the metaphase II (MII) stage of the second meiosis, a process known as oocyte maturation. These mature oocytes, henceforth referred to as eggs, are ovulated arrested at the MII stage, from which they are released at the time of fertilization. As in many other cells, Ca2+ homeostasis plays a pivotal role in oocytes during maturation and following fertilization, and it is therefore carefully regulated in these cells. Both intracellular Ca2+ release and Ca2+ influx from the external milieu are needed to support oocyte maturation and initiation of embryo development. Ca2+ influx fills the internal Ca2+ stores among which is the endoplasmic reticulum (ER), [Ca2+]ER. During maturation, the stored Ca2+ is needed to promote protein synthesis that makes possible oocyte maturation as well as embryo development. Further, the release of this Ca2+ that causes repeated changes in the intracellular concentration of free calcium ([Ca2+]i) also known as oscillations is required for the release of the MII arrest, which leads to egg activation and initiation of embryo development; Ca2+ influx is required to support these oscillations. Multiple plasma membrane (PM) channels in oocytes and eggs have been identified, including voltage dependent ion channels and members of the Transient Receptor Potential (TRP) family that are thought to mediate Ca2+ influx and the influx of other divalent cations. However, the extent of activity and regulation of these channel(s) during maturation and fertilization are unknown. Furthermore, the fertilization induced Ca2+ oscillations in mammals induce a sequence of biochemical and phenotypical changes in the egg that cause egg activation and zygote development. Some of these changes include cortical granule exocytosis, cell cycle progression and release of the second polar body, sperm head decondensation, pronuclear formation and DNA synthesis and eventually cleavage to the two-cell stage. Many of these events are mediated by a Ca2+ dependent Calmodulin (CaM)-dependent kinase, (CaMKII). CaMKII transduces Ca2+ oscillation parameters such as amplitude and frequency into changes of enzyme activity, which have been investigated in eggs using in vitro assays. Nevertheless, the pattern of CaMKII activity throughout the [Ca2+]i oscillations in real time has not been described in eggs nor what aspects of the Ca2+ rise parameters, amplitude and/or duration are important for the activation of the enzyme. Hence, understanding the molecular basis of Ca2+ homeostasis and in this particular case the channels that mediate divalent cation influx during maturation, fertilization and initiation of embryo development, as well as the parameters that activate the key enzyme that transduces [Ca2+]i rises into events of egg activation will provide pivotal insights into the mechanisms of oocyte maturation, egg activation and embryo development. The ultimate goal is to use this knowledge to aid in the treatment of infertility in humans, enhance reproductive efficiency in animals, and design novel strategies of contraception to be used in humans and to control animal populations.

Available for download on Saturday, May 08, 2021

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