|Thomas R. Peavy|
My research focus is on the molecular mechanisms that mediate sperm and egg interactions during fertilization in vertebrates. Fertilization is a highly choreographed event that is essential for species survival. Regulation of the fusion of a single sperm pronucleus (1N) with the egg pronucleus (1N) in forming the zygote (2N) is initially mediated at the extracellular matrix of the egg. I am interested in understanding how the molecular constituents of the egg extracellular matrix (egg envelope) facilitate successful species-specific fertilization, and how their structure and function have evolved in different vertebrate species. The answers to these questions have potential application towards clinical diagnoses of infertile couples, assisted reproductive technologies, contraceptive strategies, and conservation biology.
To address these questions, I use vertebrate model systems (i.e. frogs, chicken,
pig, mice, monkey) and a combination of molecular, cell, and biochemical approaches
(e.g. PCR, sequence analysis, bioinformatics, microscopy, in vitro fertilization,
protein purification, etc.). Anuran amphibians (frogs) are my preferred model
system for studies because they have several important advantages over mammalian
systems. Frogs supply large amounts of gamete material for biochemical analyses
and functional studies whereas mammals generally provide only a few eggs per
ovulation cycle. In addition, fertilization for frogs occurs externally (usually
in pond water) making experimental manipulation more accessible. In contrast,
mammalian fertilization occurs internally in the oviduct. Futhermore, amphibians
are pivotal vertebrate organisms for evolutionary and developmental studies
since they transitioned from water to land and many of their molecular adaptations
have been preserved. My central hypothesis is that by studying the molecular
structure, function and evolution of amphibian egg envelope genes, we can utilize
this information for comparative evolutionary analyses to further understand
the structure and function of vertebrate egg envelope genes.
Structure and Function of the Egg Envelope
The vertebrate egg envelope is a thick glycoproteinaceous extracellular matrix structure that surrounds the egg plasma membrane termed the chorion in fish, vitelline envelope in amphibians, and zona pellucida in mammals. These vertebrate egg envelope structures are comprised of homologous gene products referred to as ZP genes (nomenclature derived from Zona Pellucida). One of the major functions of the egg envelope is to prevent (or limit) sperm access directly to the egg’s plasma membrane. In addition to serving as a penetration barrier, the egg envelope mediates sperm recognition and contact by a “lock and key” binding interaction between sperm surface molecules and egg envelope glycoproteins. After a single sperm fuses with the egg, the egg envelope then functions (in most species) to prevent further binding and penetration of sperm so that abnormal fertilization by more than one sperm (polyspermy) does not occur. This block to polyspermy is accomplished by modification of the egg envelope constituents. After fertilization, the egg envelope becomes “hardened” and serves to protect the embryo during early development. The egg envelope is then shed as the embryo hatches from it to either implant into the uterus or to feed.
Sperm-binding components of the egg envelope
I have been examining the egg envelope ligands for sperm-binding receptors because this is a key regulatory step in the fertilization process. Recently, it has come to light that genes involved in fertilization are one of the most rapidly evolving group of proteins, a process that is thought to drive speciation events beneficial for species survival (Swanson et al., 2001). The egg envelope ZP genes (e.g. ZPA, ZPB and ZPC), are included in this group, where to date the few studies that have been performed have focused on the sperm binding ligands ZPA and ZPC for a few mammalian species. I have been interested in how deeply this phenomenon of rapid ZP evolution has influenced vertebrate speciation, which amino acid sites are rapidly evolving, how the ZP gene families are related to each other, and how these ZP components have diversified structurally and functionally.
Moreover, duplication of ZP genes (paralog gene formation) is likely to have influenced their functional diversification and potentially sperm-binding capabilities. My analyses of egg envelopes and cDNA cloning from five frog species have revealed that multiple ZPC gene products are expressed in their envelopes, suggesting that the ZPC gene has been duplicated a multitude of times in amphibians. This has led to some very intriguing questions regarding the duplication and divergence of vertebrate egg envelope genes and functional evolution. Do multiple ZPC genes exist in vertebrate lineages after the split of amphibians? If so, what is their relationship to the other ZPC genes (i.e. orthologous or paralogous)? Are amphibian ZPC genes subjected to positive Darwinian selection (high rates of mutations that result in frequent amino acid substitutions) similar to mammalian ZPC genes? To answer these questions, molecular evolutionary analyses will be performed on PCR amplified ZPC genes from cDNA libraries and also on genes identified by bioinformatics approaches (i.e. genome and protein database mining). The answers to these questions will help us to understand how deeply rooted the duplication and diversification of sperm receptor ligands has influenced gamete interactions and vertebrate speciation. I anticipate that this data will lead to the development of new hypotheses that can be tested (e.g. sites important for sperm binding).
Cortical granule exocytosis and the lectin-ligand block to polyspermy
The block to polyspermy is essential since the fusion of multiple sperm with an oocyte is pathological in most species. Approximately 1% of natural human conceptions and about 10% of IVF trials result in polyspermic fertilizations (Wessel et al., 2001). Not much is known about the molecular mechanisms that are responsible for the block to polyspermy in mammals, however studies using the model vertebrate X. laevis have been very informative. In the African clawed frog Xenopus laevis, a calcium-dependent galactosyl-specific lectin (lectin=protein that has binding sites for specific carbohydrates on molecules) from the egg cortical granules was shown to be exocytotically released upon egg activation, bound to ligands at the outer edge of the vitelline envelope, and functioned in the block to polyspermy (Greve and Hedrick, 1978; Grey et al., 1976; Wyrick et al., 1974). I have since shown that a cortical granule homolog of the lectin is found in mammals and functions to prevent sperm penetration also (Peavy and Hedrick, manuscript in preparation).
Further studies are necessary to understand the molecular mechanism of this evolutionarily conserved lectin-ligand block to polyspermy. One of the burning questions is what the molecular identity of the ligand is in vertebrate eggs. Additionally, I am interested in determining the DNA-binding elements and transcription factors that regulate developmentally-dependent expression. Other cortical granule constituents may also be coordinately regulated by these transcription factors. As it turns out, this cortical granule lectin gene is expressed in a variety of other tissues as shown in X. laevis (Chang et al., 2003), mice and humans (Tsuji et al., 2001; Suzuki et al., 2001), and therefore likely has more than one role during development. Their functional role at these different locations is only speculative but may be involved in cell-cell interactions and even host defense. Virtually nothing is known about their ligand partners. Thus, these studies on the expression of the cortical granule lectin and identification of the ligand will impact fertilization biology as well as several other fields.