Date of Award:

8-2012

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Animal, Dairy, and Veterinary Sciences

Advisor/Chair:

Dr. Kenneth L. White

Abstract

The objectives of this research were to better characterize the protein signaling complexes that form in response to spermatozoa binding to the bovine oocyte vitelline membrane and to elucidate their potential involvement in oocyte activation.

Integrins located on the vitelline membrane of bovine oocytes have been implicated in mediating the sperm-oocyte interaction. Anti-integrin function blocking antibodies and immunofluorescence were utilized in order to reveal that the αV and β1 integrin subunits are essential for fertilization in the bovine and could form the integrin heterodimer involved in the sperm-oocyte interaction.

Focal adhesion kinase is localized to focal adhesions and is a key component of signal transduction pathways mediated by integrins. The presence of focal adhesion kinase in bovine oocytes was verified by real-time polymerase chain reaction and immunoprecipitation and the localization of focal adhesion kinase at the site of sperm binding to the oocyte plasma membrane was verified using immunohistochemistry. The inhibition of focal adhesion kinase resulted in fewer cleaved embryos in addition to a reduction in the number of oocytes responding with calcium transients.

Phospholipase C isoforms regulate the release of calcium from the endoplasmic reticulum and are known to interact with integrins and focal adhesion kinase. The experiments reported in this dissertation explored the involvement of phospholipase C isoforms endogenous to the oocyte in mediating the calcium release associated with fertilization. Reduction in phospholipase C messenger ribonucleic acid levels for the phospholipase C isoforms γ1 and γ2 resulted in significantly lower cleavage rates compared to the controls. Interestingly, the reduction in messenger ribonucleic acid levels for phospholipase ζ failed to impact cleavage. Maximizing protein levels for the phospholipase C isoforms ζ and γ2 resulted in a significantly higher number of oocytes reaching the 2-cell stage compared to all other treatment groups and not significantly different than the activation control. Together these data illustrate the involvement of the αV and β1 integrin subunits, focal adhesion kinase, and the potential involvement of multiple endogenous phospholipase C isoforms (γ1 and γ2) in bovine oocyte activation. A more complete understanding of the molecular players involved in fertilization could have beneficial impacts for human fertility, assisted reproduction, and improved efficiency of animal somatic cell nuclear transfer.

Comments

This work made publicly available electronically on September 20, 2012.

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