Investigation of the levels of sperm-binding gene expression in frogs from the genera Xenopus and Lepidobatrachus

Thesis (M.S., Biological Sciences (Molecular and Cellular Biology)) -- California State University, Sacramento, 2011.Pre-zygotic blocks to fertilization create scenarios where two members of an interbreeding population are unable to conceive and produce offspring resulting in reproductive isolation. This inability to naturally produce offspring could be a result of the failure of sperm binding to the exterior of the female egg. The mammalian oocyte is surrounded by an extracellular matrix termed the zona pellucida (vitelline envelope in amphibians) that houses glycoproteins (ZPC) that function to bind sperm in a species-specific manner to prevent hybridization. Zebrafish have experienced numerous ZPC gene duplications creating multiple copies within the genome, albeit non-functional with regards to sperm-binding. On the other hand, mammals have only one ZPC gene copy that codes for and functions as the primary sperm-binding protein. Evolutionarily between these two groups of taxa lie amphibians in which ZPC is also a sperm-binding protein. Multiple gene copies have been found to be expressed in three species of frogs: Xenopus laevis, Xenopus borealis, and Lepidobatrachus laevis. The discovery of multiple ZPC genes has led to further questions as to why multiple copies are expressed and to what degree. Since determining the function of each ZPC gene would require considerable effort (purified proteins and sperm binding assays), the focus of these studies is on the expression level of each gene because it is likely to be informative as to the functional role the translated protein plays in fertilization. One possibility is a single ZPC copy is expressed in higher amounts likely rendering it the functional sperm-binding gene while remaining gene copies play structural roles within the vitelline envelope. Alternatively, all ZPC genes may be able to bind sperm, and expression of these ZPCs in the same egg would cause competition for sperm-binding. Competition may have led to the evolution of binding affinity differences where the gene product with the greatest affinity for sperm will have a decided advantage for binding. However, the situation could be a combination of the first two scenarios where multiple genes expressed in high amounts perform sperm-binding while remaining low expressing gene copies perform alternative roles. My hypothesis is that Xenopus and Lepidobatrachus ZPC genes are unequally expressed within their respective ovaries and that orthologous genes show a similar pattern of expression. Levels of gene expression were determined from ovary cDNA libraries using quantitative PCR (qPCR) which measures the expression of each ZPC gene as compared to a reference gene. The reference gene, in this case GAPDH, is used as a baseline to which all qPCR expression data is normalized. Using qPCR, the ZPC gene expression profiles were determined for individuals from two closely related species X. laevis and X. borealis as well as the distant relative L. laevis to investigate if gene copies are expressed in higher amounts relative to others. Through qPCR and ANOVA statistical hypothesis tests, ZPC genes were found to be unequally expressed in Xenopus and Lepidobatrachus. Furthermore, phylogenetic evidence suggests X. laevis and X. borealis ZPC orthologs have maintained a relatively similar level of expression after their ancestral split. Genes with comparable expression levels may share a common role with predominantly expressed genes performing as functional sperm-binding proteins while low expressers may have lost their ability to bind sperm. While precise functional roles of each ZPC cannot be assigned from this work, the discovery of unequal expression provides preliminary information for future experimental design aimed at elucidating the functions of the different ZPC genes.Biological Sciences (Molecular and Cellular Biology