MCDB Home > Faculty > Kathleen Foltz > Current Research

Research in our laboratory is focused on elucidating the molecular mechanisms of oocyte maturation, gamete recognition, and subsequent egg activation. After contact of the sperm with the proper egg, the two cells fuse, the fertilized egg is "activated" and the developmental pathway is initiated. We use many species of marine invertebrates, such as sea urchins, sea stars and ascidians, as model systems to address these phenomena. Both large-scale biochemical approaches as well as single cell (microinjection and microscopic imaging) experiments can be conducted easily using the eggs of these free spawning animals, which share the basic aspects of egg activation with other species, including mammals. Completion of the sea urchin (Strongylocentrotus purpuratus) genome project has opened up new lines of inquiry, which we are incorporating into our research plans. Our group serves as a designated genome annotation sub group and we are engaged in cataloging and describing the transcripts and proteins expressed in the sea urchin egg, focusing on functional aspects. Brief summaries of some of our current research are provided below.

How does sperm-egg interaction trigger release of Ca²⁺ in the egg?

The activation of the quiescent egg during fertilization is the remarkable first step in the development of a new organism. Despite a long history of descriptions of the process, surprisingly little is known about the molecular details of fertilization in any system. In the eggs of all multicellular animals studied to date, a rise in intracellular Ca²⁺ plays a key role in egg activation. How is this Ca²⁺ rise mediated? In echinoderm (sea urchin and sea star), ascidian, and perhaps in some vertebrate eggs (fish, frog), sperm somehow trigger activation of a Src Family Kinase (SFK), a non-receptor tyrosine kinase, which in turn activates a phospholipase (PLCg) enzyme to cause production of the small molecule, inositol trisphosphate (IP₃). This IP₃ then causes opening of Ca²⁺ gates in the egg's endoplasmic reticulum (ER), and thus cytoplasmic Ca²⁺ levels rise - all of this occurring in less than 1 minute after sperm-egg interaction. The Ca²⁺ is necessary and sufficient for many egg activation events, including the permanent block to polyspermy, DNA synthesis, and first cleavage. We are conducting experiments designed to test hypotheses about the molecular mechanisms of the very early events of egg activation. How are the SFKs regulated? Do they have multiple roles in early activation? What are the target proteins of the SFKs? Echinoderm and ascidian eggs, which are available in large quantity, easy to manipulate and to microinject, and which are exquisitely synchronous in their response to sperm, are being used to investigate (i) the role of SFKs in various egg activation events; (ii) the physical and functional interactions of SFKs with upstream regulators; and (iii) the identities and roles of other proteins that interact with SFKs to orchestrate the events of egg activation. We are identifying the components of the pathways as well as their organization and regulation at fertilization. At the egg plasma membrane, it appears that there are specialized membrane microdomains that serve to scaffold and organize various signaling components such as the Src family kinases and PLC-gamma; these regions may aid in forming a sort of "fertilization synapse" where the sperm and egg meet. Ultimately, we hope to identify egg surface components and sperm components that may function as the recognition and membrane fusion machines and serve as initial triggers in egg activation.

Because the rise in intracellular calcium is conserved across metazoans and because it is absolutely required to activate development, any information gained about the molecular mechanism of fertilization is useful. The echinoderms represent the best-understood model system currently in place for dissecting the egg activation pathway. In addition to the basic fertilization biology is the phenomenon of the signal transduction "switch" - the molecular control of cellular decision making. Eggs (especially the highly synchronous echinoderm eggs) offer an exciting example of digital signaling, based on protein-protein interactions, which toggles the cell from "off" to "on" in seconds. Thus, understanding the details of fertilization will provide insight into the general phenomenon of signal transduction as well.

How is the cell cycle regulated in the transition from unfertilized egg to developing embryo?

At fertilization, the calcium rise is absolutely required to re-initiate the cell cycle and launch the newly fertilized egg into the developmental program. Understanding how cells are thrust out of a quiescent (arrested) G_o state and into an active cell cycle is key to our understanding of a number of fundamental biological phenomena including cancer, stem cell biology and fertilization biology. We are engaged in experiments designed to evaluate the mechanism of entry from G_o into the first S phase of the mitotic cell cycle. Fertilization causes arrested oocytes to undergo rapid and precisely regulated mobilization of a myriad of cell activities. A key aspect of oocyte cell cycle regulation is the Extracellular signal Regulated Kinase/Mitogen Activated Protein Kinase (ERK/MAPK) signaling module. Interestingly, in contrast to the typical role it plays in somatic cells, active MAPK signaling in oocytes acts as a repressor rather than activator of cell cycle progression. Most oocytes must first finish meiosis before entering the first mitotic cell cycle, and many of the activities, including the MAPK module, that are required for meiotic completion are also involved in regulating first mitosis. The sea urchin, however, is an optimal model in which to study the events that are exclusively linked to entry into the first S phase of the cell cycle - the eggs of this animal naturally complete meiosis prior to fertilization and the haploid egg is arrested in a quiescent G_o state. MAPK signaling is required to maintain the arrest. Within seconds of fertilization, Ca²⁺ is released from intracellular egg stores, which leads to inactivation of the MAPK signal transduction module. This in turn signals transcriptional and translational-independent entry into the first S phase and progression through the first mitotic cell cycle. However, the molecular mechanisms underlying the regulation of MAPK and its action in the egg are unknown. The objectives of our research are to elucidate these mechanisms by: (1) determining how MAPK is regulated at fertilization, including (2) identifying and the phosphatase that inactivates it and the upstream regulator (Mos) that activates it; (3) identifying targets of MAPK using a functional proteomics approach; and (4) determining the roles of the targets in the G_o /S transition. Successful completion of these Aims will provide insight into how MAPK signaling regulates mitotic cell cycle entry in arrested eggs, which has implications not only for mechanisms of basic cell cycle regulation, but also for egg activation at fertilization.

How is oocyte maturation regulated?

This project has grown out of an international collaboration with Dr. Andrew Giusti, Dr. Laurinda Jaffe and labs in Japan, Chile and Canada through the Human Frontiers in Science Program. It is focused on the identification of the oocyte membrane receptor for the sea star oocyte maturation hormone, 1-methyl adenine (1-MA). Oocyte maturation is characterized by cell cycle re-entry of an arrested oocyte to reach fertilization competence. Although this event is fundamental to development of all higher eukaryotes, the receptor for an oocyte maturation hormone has yet to be identified definitively in any species studied to date. While the key components of the maturation network in oocytes appear to be cellular signaling molecules common to most species, the key initiator in this cellular cascade, binding of maturation hormone to its cell surface receptor, continues to elude researchers. The goal of our project is to identify and clone a cDNA encoding the oocyte maturation hormone receptor for the sea star, Asterina miniata (recently re-classsified as Patiria miniata). This animal is well suited for oocyte maturation research and specifically, as a model system for identifying the maturation hormone receptor. Oocytes are easily isolated in large quantity and oocyte maturation can be conveniently reproduced in the lab. Furthermore, the starfish oocyte maturation-inducing hormone, 1-MA, was identified over 30 years ago by Kanatani and co-workers. Work over the years by a number of researchers has revealed that 1-MA stimulates oocyte maturation via a receptor located in the oocyte plasma membrane that is coupled to a G-protein-linked and PI3 kinase-linked pathway. Thus, the sea star oocyte represents a system in which the physiological hormone is identified and a number of key properties of the receptor and its signaling properties have been described. However, many researchers have tried unsuccessfully to isolate the 1-MA receptor using traditional biochemical approaches. Our apporach is designed to identify and characterize this receptor using unique homology based and functional expression cloning based approaches. We are using two distinct strategies in tandem to identify candidate cDNAs. The first strategy is based on structural homology and makes use of degenerate rtPCR based on known G-protein coupled receptor sequences. The second strategy is based on functional expression cloning using a high throughput screen in yeast. In addition to sequence analysis, cDNAs isolated in either primary screen are subjected to a secondary, function-based screen using a rapid expression assay in Xenopus oocytes, in collaboration with Dr. Rindy Jaffe's group at UCHC. Each candidate cDNA that we identify is then subjected to binding studies designed to support or reject the hypothesis that a given cDNA encodes the 1-MA receptor. If a cDNA encodes an authentic 1-MA receptor, then we predict that the protein will bind specifically to 1-MA with a k_d approximating that described for binding to oocytes. Once we have the receptor in hand, we will pursue multiple avenues of investigation to dissect the structure and function of the protein in order to gain insight into the remarkable phenomenon of oocyte maturation.

Figures and Additional Information

Figure 1

Current model for the calcium release pathway at fertilization in echinoderm eggs. Fertilization somehow results in the activation of an egg signal transduction pathway involving a Src family kinase (SFK) and PLCg, Generation of IP3 results in the opening of the IP3 receptor (a calcium gate) in the egg's endoplasmic reticulum (ER), allowing calcium entry into the egg cytoplasm. The rise in calcium results in cortical granule exocytosis as part of the slow block to polyspermy. Calcium is also necessary and sufficient to initiate DNA synthesis and activate the cell cycle.

Figure 2

A sea star (Asterina miniata) mature oocyte being microinjected using a fine glass needle. The oocyte is approximately 170 microns in diameter. Photo by A.F. Giusti.

Figure 3

Many species of local echinoderms (shown are sea urchins, sea stars and sea cucumbers) are easily collected and maintained using UCSB's state of the art seawater system. UCSB is one of the few universities with its marine laboratory located on the main campus. Photo by N.L. Adams.

Figure 4

The purple sea urchin (Strongylocentrotus purpurtaus) is a favorite model organism for studying fertilization and early development. Many gametes (sperm are white, eggs are yellow-orange) can be obtained from a single animal and fertilization and cell division are exquisitely synchronous. These animals were collected off the Goleta Pier and tested for their gamete load on the boat. Photo by S. Anderson.

Figure 5

Two cell stage sea urchin embryos, approximately 2 hours after fertilization. The two blastomeres are surrounded by the transparent, protective fertilization envelope. The embryos will continue to divide regularly and synchronously every 30 minutes or so until they reach the late blastula stage, at about 20 hours post-fertilization.