·Zygote 2 (August), pp 221-225. Copyright© 1994 Cambridge University Press
Printed in Great Britain
· Producing exposed coat-free embryos
.Michael F. Daily, Virginia H. Latham, Claudia M. Garcia, Cynthia L. Hockman, Helen Chun, Mark
L. Oppenheimer, Steven P. West, Karolin Rostamiany, Richard L.C. Chao, Edward G. Pollock and
Steven B. Oppenheimer
Department of Biology and Center for Cancer and Developmental Biology, California State University, Northridge,
California, USA
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Summary
Production of embryos that are free of tough outer coats facilitates studies that arc not possible with
embryos surrounded by impene trable envelopes. This report describes a new procedure for preventing
formation of fertilisation membranes in the sea urchin (Lytechinus pictus) model. This procedure
involves treating unfertilised eggs with the enzyme alpha-amylase, w hich cleaves alpha-1,4 glucosidic
bonds in the vitelline layer. A major advantage of this method is that it is very well defined and
completely controllable with alpha-amylase inhibitor. The results suggest that intact alpha-1,4 glucosidic bonds are essential for vitelline layer integrity required for formation of the fertilisation membrane. Eggs treated with alpha-amylase possessed the same surface lectin receptors as untreated eggs
and, as shown by light and transmission electron microscopy, produced healthy, cleaving embryos that
were free of fertilisation envelopes.
Keywords: Fertilisation membrane, Sea urchin, Vitelline
Introduction
Removal of surface coats from eggs and embryos
allows study of the exposed plasma membrane and
facilitates experimental manipulations during early
development. The sea urchin egg is a model for such
studies. It is available by the billions and early embryonic development is easily observed under the simplest
of conditions (reviewed in Giudice, 1986;. Oppenheimer & Lefevre, 1989).
The vitelline layer, which lies outside the plasma
membrane of the unfertilised egg, consists of several
proteins ranging in size from 25 to 213 kDa and contains 3.5% sugar (fucose, mannose, galactose, glucose,
xylose, glucosamine, galactosamine and sialic acid:
Giudice, 1986). Upon fertilisation, the vitelline layer
together with material from cortical granules forms
the f~rtilisation membrane, a tough outer .coat that
prevents entry of additional sperm and makes the
embryo impenetrable to exp erimental manipulations.
All correspondence to: Steven B. Oppenheimer, De partment
of Biology and Center for Ca ncer and Devc.loplllental .
Biology, California Stilte University, Northridge, 18111
N ordhoff Street, Northddgc, CA 91330-8303, USA. Tel: 818/
885-3336. Fax: 818/717-4030.
In this study, we describe a new method for preventing fertilisation membranes, compare it with
previous methods and discuss its usefulness for
experiments that require coat-free embryos.
Materials and methods
Sea urchins (Lytechinus pictus) were purchased from
Marinus, Inc. (Long Beach, CA) and maintained in
refrigerated aquaria at 10°C. Gametes were obtained
using standard methods by inoculating adult urchins
with 1-2 ml of 0.55 M KCI. Extruded eggs were collected and washed in artificial seawater (ASW), pH 8 .0,
and stored on ice. Sperm was stored undiluted on ice
in 100 x 15 mm plastic Petri plates. Alpha-amylase ( x 4
crystallised, -catalogue number A-6380), alpha-amylase
inhibitor (catalogue number A-3410), dithiothreitol
(OTT), lectin derivatised agaxose beads, succinyl
concanavalin A a~d alpha-methylglucose were
obtained from Sigma (StLouis, MO).
Eggs for all experiments were dejellied in pH 4.0
ASW at 15 ac and returned to pH 8.0 ASW. Egg vitelline layers were kept intact, or altered by a standard
222
M.F. Daily et al.
method using DIT or with the method developed
here using alpha-amylase. The DIT procedure
involved gently swirling 2 ml of eggs in 10 ml of
0.02 M DTT in pH 9.1 ASW for 4 min at 20°C. The
volume was brought to 200 ml with pH 8.0 ASW. After
the eggs settled, the supernatant was aspirated off and
100 ml of pH 8.0 ASW were added. Eggs were allowed
to settle once again, the seawater removed and 50 ml
of pH 8.0 ASW were added (Epel et al., 1970; Carroll et
al., 1977).
For alpha-amylase treatment, each millilitre of eggs
was incubated with 200 units alpha-amylase for
30 min at 20°C in pH 8.0 ASW. Treated eggs were
washed three times with pH 8.0 ASW. In some experiments 700 units of alpha-amylase inhibitor were
added to the alpha-amylase before addition of eggs. In
some experiments, untreated, DTT-treated and alphaamylase-treated eggs were fertilised by adding 0.1 ml
of diluted sperm (0.1 ml dry sperm in 1 ml ASW,
pH 8.0) to 1 ml eggs in pH 8.0 ASW at 15 oc_ The sperm
concentration was chosen to ensure maximum fertilisation in experimentals and controls.
Untreated, DTT-treated or alpha-amylase-treated
unfertilised eggs were rotated for 1 h at 45 rpm in
pH 8.0 ASW at JSOC with various lectin derivatised
agarose beads to obtain some idea of the nature of
lectin receptors on the surfaces of these eggs. In other
experiments, treated and untreated eggs were incubated with 0.04 mg succinyl concanavalin A per millilitre, with or without 0.1 M alpha-methylglucose in
pH 8.0 ASW, prior to· fertilisation. In all experiments
results were videotaped u sing a Panasonic WW 1504X
video camera attached to a Leitz inverted microscope.
Photographs were made from videotape with a Polaroid Freezeframe Plus Video Image Recorder.
Treated and untreated unfertilised and fertilised
eggs were fixed for 3 h at room temperature in
Karnovsky' s fixative diluted 1:1 with 0.2 M cacodylate
buffer (pH 7.4) as in Kawabe et al. (1981). The eggs
were washed in 0.2 M cacodylate buffer (pH 7.4) four
times, and postfixed in 1% osmium tetroxide in 0.1 M
cacodylate buffer (pH 7.4). The eggs were washed
three times with 0.2 M cacodylate buffer (pH 7.4),
dehydrated in ace tone and embedded in Spurr's low
viscosity epoxy resin. Thin sections were cut with a
Reichert ultramicrotome, stained with uranyl acetate
and lead citrate, and examined with a Zeiss EM-10
transmission electron microscope at 60 keV.
Results
Eggs treated with alpha-amylase, followed by addition of sperm, were fertilised and cleaved, but did not
p ossess fertilisation membran es. Identical results were
obtained with the standard OTf method. Alpha-
amylase inhibitor blocked the effect of alpha-amylase
in preventing formation of fertilisation membranes.
Untreated, OTT-treated and alpha-amylase-treated
eggs in 3-13 separate experiments with .each type of
lectin d erivatised agarose bead (preferential binding
sugars in parentheses) bound well to beads derivatised with Dolichos biflorus (N-acetyl-o-galactosamine),
nearly as well to beads d erivatised with concanavaliln
A (alpha-o-mannose, alpha-o-glucose), but did not
bind at all to beads derivatised with Vicia villosa
(N-acetyl-o-galactosamine), Pisum sativum (alpha-omannOSE!), Ulex europaeus (alpha-L-fucose), Phytolacca
americana [(N-acetyl-o-glucosamineh], Lens culinaris
(alpha-o-mannose), or Tetragonolobus purpureus
(alpha-L-fucose}. Fertilisation of untreated, DITtreated and alpha-amylase-treated eggs was inhibited
by 0.04 mg succinyl concanavalin A per millilitre, an
effect that was blocked by OJ M alpha-methylglucose.
The electron micrographs in Figs. 1-4 show that
alpha-amylase removes some vitelline layer material
from eggs (Figs. 1, 2), and that no fertilisation m embranes form when these eggs are fertilised (Figs. 3, 4).
Identical results were obtained· using the standard
DTT procedure.
Discussion
Visual observations indicate that eggs treated with
alpha-amylase behave like untreated eggs or eggs
treated with the standard DTI procedure. All three
groups of eggs cleave normally and bind to the same
lectin derivatised agarose beads, and their fertilisation
is inhibited by succinyl concanavalin A, a n effect
blocked by alpha-methylglucose. These results
suggest that alpha-amylase treatment and DTT
treatment preserve egg surface lectin receptors that
may play a role in sperm-egg interaction.
A variety of methods has been d eveloped to
prevent formation of fertilisation membranes in sea
urchin eggs. Some involve proteases, which can
remove plasma membrane protein as well as vitelline
layer; o thers u se OTT and protease; while still others
use para-chloromercuribenzoate or 1M urea (Epel et
al., 1970; Carroll eta/., 1977; BerSt 1967; Schatten &
Schatten, 1979). Some of these methods are quite
harsh, involve time-consuming procedures, require
large volumes of material, or are very sensitive to pH
or temperature. All of them involve procedures the
mode of action of which is not well understood. If a
major requirement of a p articular experiment is to
remove the vitelline layer completely, then the procedure of Carroll et al. (1977) is the method of choice,
because they definitively d emonstrated that their procedure, utilising natural cortical granule protease plus
OTT, completely removed the vitelline layer.
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Producing exposed coat-free embryos
This laboratory has for many years been involved in
studying the role of surface sugars in a variety of cell
recognition events. Alpha-amylase was originally
used in the laboratory to examine its effect on spermegg interaction. At first it was assumed that because no
fertilisation membranes formed, the degradation of
egg surface polysaccharide was removing sperm
receptors preventing fertilisation. This, however, was
not the case because the eggs were fertilised and
cleaved but did not form fertilisation envelopes. Since
the procedure was simple, specific, well-defined and
controllable with alpha-amylase inhibitor, it was
decided to develop it as an improved method for
producing embryos free of fertilisation membranes.
Since alpha-amylase action in this system is completely blocked with alpha-amylase inhibitor, it is
likely that the observed effects of this enzyme result
from its known specific action, rather than from some
impurity in the 4 times crystallised enzyme
preparation.
Alpha-amylase hydrolyses alpha-1,4 glucosidic
bonds in polyglucosans (amylose, amylopectin, glycogen and dextrins) (Bernfeld, 1951; Chung & Friedberg,
1980). The vitelline layer consists of protein and sugar,
but it is incompletely characterised. This study suggests that alpha-1,4 glucosidic bonds are essential for
vitelline layer integrity and for its ability to form the
fertilisation membrane. This method, unlike many
others described, has a specific known mode of action,
which is controllable with alpha-amylase inhibitor
(Silano eta/., 1975; O'Donnell & McGeeney, 1976). It
may be the method of choice for experiments where it
is essential to know how the reagent used ac~s. It may
also be a method of choice for experiments where
protease or DIT cannot be used.
Because of the specificity of this enzyme's action,
further exploration of its effects on the vitelline layer
may improve our understanding of the layer's architecture and its involvement in forming the fertilisation
membrane. This simple procedure facilitates the rapid
production of healthy coat-free embryos that can be
used in a variety of experimental studies.
225
Acknowledgements
This work was supported by grants from NIH MARC,
NIH MBRS, NSF-TPE, LAEP-IISME, the Thomas
Eckstrom Trust and the Joseph Drown Foundation.
References
Bernfeld, P. (1951). Enzymes of starch degradation and synthesis. Adv. Enzymol. 12, 379-428.
Berg, W.E. (1967). Some experimental techniques for eggs
and embryos of marine invertebrates. In: Methods in
Developmental Biology, ed. F.H. Wilt & N.K. WesseHs,
pp. 767-76. New York: Crowell.
Carroll, E_J., Byrd, E.W. & Epel, D. (1977). A novel procedure
for obtaining denuded sea urchin eggs and observations
on the role of vitelline layer in sperm reception and egg
activation. Exp. Cell Res. 108, 365-74.
Chung, H. & Friedberg, F. (1980). Sequence of the Nterminal half of Bad/lis amyloliljuefaciens alpha amylase.
Biochem. /. 185, 387-95.
Epel, D., Weaver, A.M. & Mazia, D. (1970). Methods for
removal of the vitelline membrane of sea urchin eggs. I.
Use of dithiothreitol (Cleland reagent). Exp. Cell Res. 61,
64-S.
Giudice, G. (1986). The Sea Urchin Embryo. Berlin: Springer.
Kawabe, T.K., Armstrong, P.B. & Pollock, E.G. (1981). An
extracellular fibrillar matrix in gastrulating sea urchin
embryos. Dev. Bioi. 85, 509-15.
O'Donnell, M.D. & McGeeney, K.F. (1976). Purification and
properties of an alpha amylase inhibitor from wheat.
Biochim. Biophys. Acta 422, 159-69.
Oppenheimer, S.B. & Lefevre, .G. (1989). Introduction to
Embryonic Development, 3rd edn, pp. 84-113. Boston: Allyn
and Bacon.
Schatten, G. & Schatten, H . (1979). Sperm-egg membrane
fusions and interactions in denuded sea urchin eggs. Scanning Electron Microsc. 111, 299-305.
Silano, V., Furia, M., Gianfreda, L., Macri, A., Palescandolo,
R.A., Scardi, V., Stella, E. & Valfre, F. (1975). Inhibition of
amylases from different origins by albumins from the
wheat kernel. Biochim. Biophys. Acta 422, 159-69.