David J. Carroll

Requirement of a Src Family Kinase for Initiating Calcium Release at Fertilization in Starfish Eggs

Signal transduction leading to calcium release in echinoderm eggs at fertilization requires phospholipase Cγ-mediated production of inositol trisphosphate (IP3), indicating that a tyrosine kinase is a likely upstream regulator. Because previous work has shown a fertilization-dependent association between the Src homology 2 (SH2) domains of phospholipase Cγ and a Src family kinase, we examined whether a Src family kinase was required for Ca2+ release at fertilization. To inhibit the function of kinases in this family, we injected starfish eggs with the SH2 domains of Src and Fyn kinases. This inhibited Ca2+ release in response to fertilization but not in response to injection of IP3. We further established the specificity of the inhibition by showing that the SH2 domains of several other tyrosine kinases (Abl, Syk, and ZAP-70), and the SH3 domain of Src, were not inhibitory. Also, a point-mutated Src SH2 domain, which has reduced affinity for phosphotyrosine, was a correspondingly less effective inhibitor of fertilization-induced Ca2+ release. These results indicate that a Src family kinase, by way of its SH2 domain, links sperm-egg interaction to IP3-mediated Ca2+ release at fertilization in starfish eggs.

Protein tyrosine kinase signaling during oocyte maturation and fertilization.

The oocyte is a highly specialized cell capable of accumulating and storing energy supplies as well as maternal transcripts and pre‐positioned signal transduction components needed for zygotic development, undergoing meiosis under control of paracrine signals from the follicle, fusing with a single sperm during fertilization, and zygotic development. The oocyte accomplishes this diverse series of events by establishing an array of signal transduction pathway components that include a select collection of protein tyrosine kinases (PTKs) that are expressed at levels significantly higher than most other cell types. This array of PTKs includes cytosolic kinases such as SRC‐family PTKs (FYN and YES), and FAK kinases, as well as FER. These kinases typically exhibit distinct patterns of localization and in some cases are translocated from one subcellular compartment to another during meiosis. Significant differences exist in the extent to which PTK‐mediated pathways are used by oocytes from species that fertilize externally versus internally. The PTK activation profiles as well as calcium signaling pattern seems to correlate with the extent to which a rapid block to polyspermy is required by the biology of each species. Suppression of each of the SRC‐family PTKs as well as FER kinase results in failure of meiotic maturation or zygote development, indicating that these PTKs are important for oocyte quality and developmental potential. Future studies will hopefully reveal the extent to which these factors impact clinical assisted reproductive techniques in domestic animals and humans. Mol. Reprod. Dev. 78:831–845, 2011.

Combining Microinjection and Immunoblotting to Analyze MAP Kinase Phosphorylation in Single Starfish Oocytes and Eggs

The starfish oocyte has proven useful for studies involving microinjection because it is relatively large (190 mum) and optically clear. These oocytes are easily obtained from the ovary arrested at prophase of meiosis I, making them useful as a model system for the study of cell cycle-related events. In this chapter, a method for combining microinjection with immunoblotting of single cells is described. Individual starfish oocytes are injected, removed from the microinjection chamber, and analyzed by immunoblotting for the dual-phosphorylated form of mitogen-activated protein kinase (MAPK). This method will allow for experiments testing the regulation of MAPK in single cells and for the manipulation of these cells by a quantitative microinjection technique.

Signal transduction during fertilization: Studies with proteases and heterologous receptors

Summary Recent investigations of fertilization have indicated the importance of signaling pathways in the egg that are initiated by cell surface receptors. The evidence includes: (1) stimulation of heterologous receptors (mammalian) in starfish eggs, leading to activation of both early (e.g., Ca2+ release) and late (e.g., DNA synthesis) events of fertilization (Shilling et al., 1994); and (2) activation of eggs by external treatment with specific proteases (Carroll and Jaffe, 1995). Heterologous receptor-induced activation involves phospholipase C (PLC). This enzyme may be activated by a G-protein mediated pathway (PLC-β) or a tyrosine kinase receptor pathway (PLC-γ). There have been other mechanisms for egg activation proposed, which will not be discussed here, such as the introduction of a “cytoplasmic factor” by the sperm into the egg (Swann, 1990). We propose a multi-subunit receptor complex as the mediator of the activating signal from sperm, with properties similar to those of other receptor-mediate...

Starfish as a Model System for Analyzing Signal Transduction During Fertilization

The starfish oocyte and egg offer advantages for use as a model system for signal transduction research. Some of these have been recognized for over a century, including the ease of procuring gametes, in vitro fertilization, and culturing the embryos. New advances, particularly in genomics, have also opened up opportunities for the use of these animals. In this chapter, we give a few examples of the historical use of the starfish for research in cell biology and then describe some new areas in which we believe the starfish can contribute to our understanding of signal transduction-particularly in fertilization.

Preparing for Fertilization: Intercellular Signals for Oocyte Maturation

In the hours preceding fertilization, oocytes prepare to begin development in a process known as maturation. This includes progression of the meiotic cell cycle and development of the ability to undergo the release of calcium that activates development at fertilization. In many species, the signal for oocyte maturation acts initially on somatic cells surrounding the oocyte, rather than on the oocyte itself. This chapter concerns the intercellular signaling events by which the maturation-inducing signal travels from the somatic cells to the oocyte. We first discuss how meiotic prophase arrest is maintained in mammalian oocytes, and how luteinizing hormone (LH) action on receptors in the cells of the surrounding follicle causes meiosis to resume. The LH receptors are located exclusively in the outer granulosa cells of the follicle and signal through a Gs-linked receptor to cause a decrease in cyclic guanosine monophosphate (cGMP). The LH-induced signal propagates inwards to the oocyte by way of cGMP diffusion out of the oocyte through gap junctions. We then briefly discuss the similarities and differences in mechanisms controlling oocyte maturation in animals other than mammals, focusing on hydrozoan jellyfish to emphasize the early evolutionary origin of these regulatory processes. G-protein-coupled receptors and cyclic nucleotides are common regulators in many animals. However, the assembly of these components into a regulatory system differs among species.

Biotinylation of oocyte cell surface proteins of the starfish Patiria miniata.

Understanding the signal transduction processes that occur during oocyte maturation and fertilization requires knowledge of the constituent proteins from the cell surface to relevant intracellular compartments. To identify starfish oocyte and egg cell surface proteins, a biotinylation method was adapted from prior protocols using B cells, leukocytes, mouse oocytes, and sea urchin eggs (Cole et al. Mol Immunol 24:699-705, 1987; Flaherty and Swann NJ. Mol Reprod Dev 35:285-292, 1993; Haley and Wessel. Dev Biol 272:191-202, 2004; Hurley et al. J Immunol Methods 85:195-202, 1985). This method utilizes the water-soluble Sulfo-NHS-Biotin, which does not cross the egg plasma membrane. The process of biotinylation does not appear to have any effect on the process of oocyte maturation or fertilization. Furthermore, it can be used with either vitelline-intact or vitelline-free oocytes and allows the proteins to be visualized successfully through immunoblotting, immunoprecipitation, or by scanning confocal microscopy.