Laboratory on Sea Urchin Fertilization
Since about 1880, the eggs and sperm of sea urchins have been used for the study of fertilization, the metabolic activation of development and gene regulatory mechanisms governing embryogenesis. Sea urchin gametes are a favorite material for observations of the process of fertilization in advanced high school, community college, and university biology laboratory courses.
This article is a laboratory handout, designed for the student to follow in learning about fertilization. In addition to observations of sperm–egg interaction, simple experiments are described that demonstrate some mechanisms involved in the process. The hope is that by making simple observations of fertilization, the student will gain an appreciation for the fact that successive generations of higher organisms are bridged by the fusion of egg and sperm, two very different single cells.
INTRODUCTION
Sea urchins are an ancient group (at least 450 million years old) of Class Echinoidea in the Phylum Echinodermata, with hundreds of species known in the world’s oceans. Sea urchin gametes are a favorite experimental material for the study of fertilization for the following reasons: the sexes are separate, it is easy to obtain the gametes from the adults, the gametes are highly uniform in maturity and purity when released into seawater, fertilization occurs external to the adult body and is therefore easy to manipulate, the financial expense is low, adults are easy to keep in aquaria, and several species are available at marine laboratories and from biological supply companies. There are two reference books on sea urchin embryonic development (Schroeder, 1986; Ettensohn et al., 2004), and much information is available on the Internet by using < sea urchin fertilization > as the query.
There are excellent movies on YouTube and Google on sea urchin fertilization. The genome of one sea urchin, Strongylocentrotus purpuratus, is known (http://www.spbase. org/SpBase/), and the genomes of other species are being sequenced. The genome size is 800 Mb (million), compared to the human genome of 3,200 Mb. The articles describing the sea urchin genome can be found in Science, volume 314: pp 939–962, November 10, 2006. A special issue of Developmental Biology (vol. 300: pp 1–495, 2006) is also devoted to the sea urchin genome. When injected with 0.5 M KCl (which is isosmotic with seawater) gravid adults of many sea urchin species spawn their gametes (Fig. 1). The Kþ ions depolarize a muscle on the outer surface of the alveolar ovary and testis. The sperm or eggs are forcibly extruded into surrounding seawater through five gonopores on the aboral surface (opposite side from the mouth).
Electric shock (6–10 V) can also be used to Abbreviations: Ap, Arbacia punctulata; Sp, Strongylocentrotus purpuratus; Sf, Strongylocentrotus franciscanus; Lp, Lytechinus pictus; Lv, Lytechinus variegatus; De, Dendraster excentricus; Ep, Echinarachnius parma; AR, acrosome reaction; CG, cortical granule; CaFSW, calcium free artificial seawater; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; FE, fertilization envelope; FSP, fucose sulfate polymer of egg jelly; HSW, seawater with 10 mM HEPES buffer, pH 8.0; JC, egg jelly coat; PABA, para-aminobenzoic acid; SBTI, soybean trypsin inhibitor; SDS, sodium dodecyl sulfate; VL, vitelline layer of the sea urchin egg.
Eggs of all species of Class Echinodea (sea urchins, heart urchins, and sand dollars) are unusual in that when they are spawned (released from the ovary), they have finished meiosis and have a complete nuclear envelope containing a haploid amount of DNA; they are actually in the G1-phase of the first mitotic division cycle. In almost all other animals, eggs are spawned and fertilized in a meiotic stage, and finish meiosis shortly after spawning, or after fertilization. Sea urchin eggs are metabolically (biochemically) dormant and undergo an explosive metabolic activation after fusing with a sperm. Protein kinases play key roles in egg activation (Townley et al., 2009).
In this laboratory we will observe some of the phenomena associated with sea urchin sperm–egg interaction (fertilization) and egg activation. A sea urchin egg, with its hydrated jelly coat, is shown in Figure 2. Ap adults may require an electric shock of 6–10 V to induce spawning.
As the Ap females spawn, one should collect the dark red/ purple eggs with a pipette as they are coming out of the gonopores, and dilute them into cold 100 ml of HEPESbuffered filtered seawater (HSW). This is important because chemicals coming from the surface of the adult females of this species will make the eggs unfertilizable. This is not a problem with the other species, so spawning females can be inverted on top of a 50- or 100-ml beaker filled with HSW so that the HSW is touching the gonopores. The negatively buoyant eggs immediately sink to the bottom.