Julian L. Wong

Defending the Zygote: Search for the Ancestral Animal Block to Polyspermy

Fertilization is the union of a single sperm and an egg, an event that results in a diploid embryo. Animals use many mechanisms to achieve this ratio; the most prevalent involves physically blocking the fusion of subsequent sperm. Selective pressures to maintain monospermy have resulted in an elaboration of diverse egg and sperm structures. The processes employed for monospermy are as diverse as the animals that result from this process. Yet, the fundamental molecular requirements for successful monospermic fertilization are similar, implying that animals may have a common ancestral block to polyspermy. Here, we explore this hypothesis, reviewing biochemical, molecular, and genetic discoveries that lend support to a common ancestral mechanism. We also consider the evolution of alternative or radical techniques, including physiological polyspermy, with respect to our ability to describe a parsimonious guide to fertilization.

Ca(2+) signaling occurs via second messenger release from intraorganelle synthesis sites.

Summary Cyclic ADP-ribose is an important Ca2+-mobilizing cytosolic messenger synthesized from β-NAD+ by ADP-ribosyl cyclases (ARCs). However, the focus upon ectocellular mammalian ARCs (CD38 and CD157) has led to confusion as to how extracellular enzymes generate intracellular messengers in response to stimuli. We have cloned and characterized three ARCs in the sea urchin egg and found that endogenous ARCβ and ARCγ are intracellular and located within the lumen of acidic, exocytotic vesicles, where they are optimally active. Intraorganelle ARCs are shielded from cytosolic substrate and targets by the organelle membrane, but this barrier is circumvented by nucleotide transport. We show that a β-NAD+ transporter provides ARC substrate that is converted luminally to cADPR, which, in turn, is shuttled out to the cytosol via a separate cADPR transporter. Moreover, nucleotide transport is integral to ARC activity physiologically because three transport inhibitors all inhibited the fertilization-induced Ca2+ wave that is dependent upon cADPR. This represents a novel signaling mechanism whereby an extracellular stimulus increases the concentration of a second messenger by promoting messenger transport from intraorganelle synthesis sites to the cytosol.

Major components of a sea urchin block to polyspermy are structurally and functionally conserved.

Summary One sperm fusing with one egg is requisite for successful fertilization; additional sperm fusions are lethal to the embryo. Because sperm usually outnumber eggs, evolution has selected for mechanisms that prevent this polyspermy by immediately modifying the egg extracellular matrix. We focus here on the contribution of cortical granule contents in the sea urchin block to polyspermy to begin to understand how well this process is conserved. We identified each of the major constituents of the fertilization envelope in two species of seaurchins, Strongylocentrotus purpuratus and Lytechinus variegatus, that diverged 30 to 50 million years ago. Our results show that the five major structural components of the fertilization envelope, derived from the egg cortical granules, are semiconserved. Most of these orthologs share sequence identity and encode multiple low‐density lipoprotein receptor type A repeats or CUB domains but at least two contain radically different carboxy‐terminal repeats. Using a new association assay, we also show that these major structural components are functionally conserved during fertilization envelope construction. Thus, it seems that this population of female reproductive proteins has retained functional motifs while gaining significant sequence diversity—two opposing paths that may reflect cooperativity among the proteins that compose the fertilization envelope.

Cell Surface Changes in the Egg at Fertilization

An egg changes dramatically at fertilization. These changes include its developmental potential, its physiology, its gene expression profile, and its cell surface. This review highlights the changes in the cell surface of the egg that occur in response to sperm. These changes include modifications to the extracellular matrix, to the plasma membrane, and to the secretory vesicles whose contents direct many of these events. In some species, these changes occur within minutes of fertilization, and are sufficiently dramatic so that they can be seen by the light microscope. Many of these morphological changes were documented in remarkable detail early in the 1900s by Ernest Everett Just. A recent conference in honor of his contributions stimulated this overview. We highlight the major cell surface changes that occur in echinoderms, one of Justs preferred research organisms. Mol. Reprod. Dev. 76: 942–953, 2009.

Diversity in the fertilization envelopes of echinoderms.

Cell surface changes in an egg at fertilization are essential to begin development and for protecting the zygote. Most fertilized eggs construct a barrier around themselves by modifying their original extracellular matrix. This construction usually results from calcium‐induced exocytosis of cortical granules, the contents of which in sea urchins function to form the fertilization envelope (FE), an extracellular matrix of cortical granule contents built upon a vitelline layer scaffold. Here, we examined the molecular mechanism of this process in sea stars, a close relative of the sea urchins, and analyze the evolutionary changes that likely occurred in the functionality of this structure between these two organisms. We find that the FE of sea stars is more permeable than in sea urchins, allowing diffusion of molecules in excess of 2 megadaltons. Through a proteomic and transcriptomic approach, we find that most, but not all, of the proteins present in the sea urchin envelope are present in sea stars, including SFE9, proteoliaisin, and rendezvin. The mRNAs encoding these FE proteins accumulated most densely in early oocytes, and then beginning with vitellogenesis, these mRNAs decreased in abundance to levels nearly undetectable in eggs. Antibodies to the SFE9 protein of sea stars showed that the cortical granules in sea star also accumulated most significantly in early oocytes, but different from sea urchins, they translocated to the cortex of the oocytes well before meiotic initiation. These results suggest that the preparation for cell surface changes in sea urchins has been shifted to later in oogenesis, and perhaps reflects the meiotic differences among the species—sea star oocytes are stored in prophase of meiosis and fertilized during the meiotic divisions, as in most animals, whereas sea urchins are one of the few taxons in which eggs have completed meiosis prior to fertilization.

FRAP analysis of secretory granule lipids and proteins in the sea urchin egg.

Cortical granules of the sea urchin are secreted at fertilization in response to sperm fusion. Approximately 15,000 of these vesicles are tightly tethered to the cytoplasmic face of the egg plasma membrane prior to insemination such that the vesicle-plasma membrane complex may be isolated and manipulated in vitro. Furthermore, this complex remains fusion competent and can thus be used for in vitro biochemical studies of secretion on a per-vesicle or a population scale. We document approaches to study the dynamics of membrane lipids and proteins in these secretory vesicles. Their large size (1.3-microm diameter), vast number, and ease of manipulation enable several unique approaches to study general secretion mechanisms.