Date of Award

7-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Ocean Engineering and Marine Sciences

First Advisor

David J. Carroll

Second Advisor

Andrew Palmer

Third Advisor

Alan C. Leonard

Fourth Advisor

Munevver Mine Subasi

Abstract

The conception of life, the creation of a genetically distinct being, is an event that has fascinated humanity for ages. It was only recently, at the turn of the 20th century, that the field of reproduction research reached an age of molecular mechanics, and with that, an onslaught of difficulties were encountered. The egg is a unique cell type because it contains a number of mRNA transcripts that do not express proteins necessary for the initial moments of gamete interaction or egg activation. Instead, these RNAs are stored in the egg for expression during early embryonic development. Coupled with the fact that reproductive proteins are under immense evolutionary pressure, making the development of putative proteomes challenging, elucidating the full molecular pathway at fertilization has yet to be accomplished. Multiple events occur between the sperm and egg membranes during fertilization, including sperm-egg binding, species-specific discrimination, membrane fusion, and egg activation. To date, only a handful of egg membrane proteins have been identified, and none of these are known to activate the fertilization signal transduction pathway in the egg. An excellent model organism to address how these events take place is the Pacific bat star, Patiria miniata, because it produces a large quantity of synchronized oocytes that are large, nearly transparent, and easy to work with. Additionally, we are learning that their genome is similar to humans, but with less complexity.Proteolytic activity from the sperm, and parthenogenic egg activation in the presence of trypsin, presented an appealing strategy for the identification critical membrane proteins. To determine if membrane proteins were being cleaved from the egg at fertilization, egg cell membrane proteins were labeled with biotin and a distinct increase in the presence of biotinylated proteins was observed in the seawater after fertilization. These were not present in the seawater around eggs that had been pre-treated with soybean trypsin inhibitor but were present in the seawater surrounding trypsin-treated unfertilized eggs, strongly suggesting the action of a trypsin-like protease on the egg surface at fertilization. These released protein fragments ranged in size between approximately 50 to 250 kDa, with unique bands only present after fertilization. The seawater eluate was collected from around unfertilized and fertilized eggs and subjected to tandem mass spectrometry to identify the released proteins. One of the chief problems studying the molecular mechanism of fertilization is the lack of a proteome of the unfertilized egg to use in conjunction with mass spectrometry for protein identification. To address this issue, three databases were generated in this work that characterize the membrane proteome in the sea star mature egg. These databases were built after first establishing the first mature egg transcriptome for the sea star, which is now available on NCBI (BioProject ID: 389668). The first database is a putative proteome of all transcripts predicted to have transmembrane alpha-helical sequences in the unfertilized egg. The second database specifically reports proteins that fall into five major signaling families that have been hypothesized to have some involvement in gamete interaction. The final database represents the membrane proteins currently present in the mature egg, as these are the proteins found after an unfertilized egg membrane preparation was analyzed by mass spectrometry. This database represents not only a putative proteome of the unfertilized egg, but further assists in categorizing thein silico proteome into those transcripts necessary for the unfertilized egg, and those needed for the rapid development the egg goes through prior to the activation of the zygotic genome. A comparison between all three databases identified the presence of five proteins that are present in the unfertilized egg and belong to one of the signaling families predicted to be involved at the surface. Of course, these are not the only important membrane proteins present in these databases; however, they represent proteins that have a greater chance of being involved in gamete interaction based on previous research in the field of fertilization. Therefore, one of the major goals of this work is to provide these targets for future mechanistic studies of the egg surface at fertilization.The development of the transcriptome to establish an in silico proteome also enabled the use of mass spectrometry to identify the proteins being released from the egg cell surface. Interestingly, vitellogenin was found to be a significant constituent of the seawater before fertilization and, thus, may be playing a role distinct from its classically defined function as a yolk protein. Also, two additional egg proteins were found to be almost exclusively present in the seawater only after fertilization. One may represent the band that shows up exclusively after fertilization at 80 kDa, and the other may be released as a mechanism for providing protection for the egg. Their removal from the membrane in response to fertilization, much like Juno is shed from the mouse egg after fertilization, could provide a means for reducing further sperm interaction with the fertilized egg. The release of critical binding proteins from the egg surface has been hypothesized to be an additional polyspermic block by binding extraneous sperm; work completed here shows that the sperm are not binding to any of the proteins being released from the egg. While binding superfluous sperm may not be the function of the proteins found in the seawater, their removal from the surface may still serve as an additional block to polyspermy by removing the potential for other sperm to bind to the egg surface. This also does not discount the possibility that the proteins being released are critical binding partners on the egg cell surface. The novel findings described here continue to move the field of fertilization forward by developing critical tools for discoveries as well as a refined list of potential targets for functionality in future work. In addition, the finding that the egg is releasing proteins prior to fertilization is a novel mechanism not previously described in any animal. All of this will work to continue to unravel the complex web that is fertilization.

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