Research Description

Species-specific fertilization in abalone

The marine mollusk abalone is the most well characterized animal model of fertilization. As a broadcast spawning marine invertebrate with external fertilization, both sperm and egg can be easily collected in enormous quantities for biochemical and functional characterization (e.g. more than 1 gram of the major sperm protein lysin can be purified from a single male abalone). Off the North American Pacific coast exist 7 abalone species (whose common names are often based on the color of their shells) with overlapping habitat ranges that creates substantial opportunity for hybridization; Despite hybrids being viable in the laboratory they are rarely observed in the wild, with molecular recognition between egg and sperm being one of the major factors that restricts heterospecific mating. Compared to mammalian counterparts, the abalone sperm proteome is remarkably simple and consists largely of only three proteins: lysin, sp18, and FITZAP. Lysin interacts with ZP-N domains of egg VERL to permeate the vitelline envelope (homologous to the mammalian zona pellucida, ZP). Sp18 is an ancient paralog of lysin with a similar tertiary structure despite low sequence similarity that is highly fusogenic and likely facilitates sperm-egg fusion. FITZAP is a small intrinsically disordered protein that enables packaging of lysin and sp18 inside the sperm acrosome at concentrations nearing one molar. As all three abalone sperm proteins are small and highly soluble, they are well suited for structural analysis by solution NMR. One barrier to abalone hybridization is the rapid co-evolution of sperm lysin and egg VERL which has resulted in weak but species-specific interactions between the gamete recognition molecules. On-going work in the lab is focused on characterizing the interactions between egg ZP-N domains, structural mechanisms of lysin-mediated egg coat dissolution, and the structure-function dynamics of sp18 with the oocyte plasma membrane.

Reproductive biology of plethodontid salamanders

Lungless salamanders (family Plethodontidae) are classic models of reproductive behavior and mating. Male and female salamanders perform stereotyped courtship behaviors that culminate in external sperm transfer for internal fertilization, and such behaviors include the “tail straddling walk” that has persisted for ~130 million years of salamander evolution. In the majority of plethodontid species, male salamanders deliver non-volatile protein courtship pheromones that modulate female mating behavior. These pheromones have been co-opted from numerous gene families and experienced rapid evolution, presumably in response to coevolutionary pressures from female receptors. Three pheromone types have been chemically purified and experimentally demonstrated to alter female mating behavior: PRF, PMF, and SPF. Remarkably, recent analysis by quantitative mass spectrometry identified paralogs of all three pheromone families as major sperm proteins that are hypothesized to play essential roles in salamander fertilization. As the most speciose family of salamanders (496 of 766 species), plethodontid salamanders have experienced expansive radiations throughout the North and Central American coasts where many species are divided along ecological gradients (e.g. elevation) with varying levels of hybridization and genetic introgression. Such hybrid zones provide natural experiments to explore how new reproductive barriers – and ultimately speciation – may evolve within homologous proteins found at both pre-mating (pheromone) and post-mating (sperm) molecular interactions.

Identification of new molecular interactions driving human fertilization

The biochemical underpinnings of human fertilization are largely a molecular mystery. In contrast to marine invertebrates such as abalone, the proteomic composition of mammalian sperm is substantially more complex in composition and functional redundancy, and there are only two known pairs of interacting sperm-egg proteins in humans. Rapid evolution and lineage-specific diversification of gametic proteins also limits the utility of genetic studies in rodent models for understanding human fertilization. My laboratory integrates quantitative mass spectrometry of human gametes with molecular evolutionary analyses and deep learning to identify new interacting pairs of fertilization proteins that are characterized using structural and biophysical techniques.