ISSN 1062-3604, Russian Journal of Developmental Biology, 2008, Vol. 38, No. 5, pp. 293–296. © Pleiades Publishing, Inc., 2008.
Original Russian Text © A.A. Minin, S.G. Ozerova, 2008, published in Ontogenez, 2008, Vol. 38, No. 5, pp. 362–366.
The interaction between the egg and sperm during
fertilization leads to the egg activation—triggering the
developmental program. One of the ﬁrst consequences
of this activation is the formation of the “fertilization
membrane” that prevents the penetration of excessive
sperm into the egg. In lower vertebrates that develop in
aquatic environment (ﬁsh and amphibians), the fertili-
zation membrane is also protective; it includes the outer
layer that was adjacent to the surface of the unactivated
egg, and the jelly water-rich layer originating from the
exocytosed contents of the egg cortical granules. The
cortical granule exocytosis is initiated by the sperm
entry into the egg in most groups of animals.
The activation and fertilization of ﬁsh eggs has been
considered and described in detail previously (Ginz-
burg, 1968). Fish have external fertilization, and the
activation can commonly proceed in the absence of the
sperm and is triggered by the egg entry into aquatic
environment, which minimizes the period when fertili-
zation is possible and prevents the entry of extra or for-
eign sperm into the egg. Such “spontaneous egg activa-
tion” leads to the formation of the fertilization mem-
brane, abnormal ooplasmic segregation, abnormal
cleavage, and eventually death. The detachment of the
fertilization membrane from the egg surface, which
becomes visible under normal conditions several min-
utes after the activation, is a simple and explicit marker
of successful egg activation. The intracellular signaling
processes related to the egg activation and triggering
the development are widely studied in various animals
including ﬁshes (Gilkey et al., 1999; Lee et al., 1999).
The increase in free calcium ion concentration is one of
key events among such processes. The cortical granule
exocytosis in the ﬁsh egg, which leads to the fertiliza-
tion membrane formation, is likely activated by calcium
ions and involves a cascade of events including the reor-
ganization of the active cortical layer in the egg (Ivanen-
kov et al., 1990; Becker and Hart, 1999). Lee et al.
(1999) demonstrated that spontaneous egg activation as
well as normal fertilization in ﬁsh induces a wave of
increase in calcium ion concentration. The mechanism
of the earliest local increase in calcium ion concentra-
tion in the animal part of the egg after activation in the
absence of sperm remains unclear (Lee et al., 1999),
which indicates yet unknown signaling that occurs dur-
ing this process.
A number of studies explored the process of egg
development without sperm in saline solutions of dif-
ferent composition (Hart and Yu, 1980; Lee et al., 1999;
Gilkey et al., 1999). The ovarian (or coelomic) ﬂuid is
commonly used in experiments on unfertilized eggs to
prevent activation; in this case it should not be neces-
sarily conspeciﬁc, for instance, the ovarian ﬂuid from
salmon can be used in experiments on zebraﬁsh eggs
(Corley-Smith et al., 1995). It remains unclear which
components of the ovarian ﬂuid prevent the ﬁsh egg
activation and what is the mechanism of the water acti-
The goal of this work was to elucidate the properties
of ﬁsh egg activation in water and water solutions of
different composition and to study the nature of the
inhibiting factors in the ovarian ﬂuid on this process.
Spontaneous Activation of Fish Eggs Is Abolished
by Protease Inhibitors
A. A. Minin and S. G. Ozerova
Kol’tsov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26, Moscow, 119991 Russia
Received June 19, 2007; in ﬁnal form, January 17, 2008
—During spawning, eggs of most ﬁsh species entering the aquatic environment remain fertilizable for
a relatively short period of time. This is due to the “spontaneous egg activation” giving rise to the fertilization
membrane, which prevents the penetration of excessive and foreign sperm into the egg during normal fertiliza-
tion. This work demonstrates that the fertilization membrane formation and the loss of fertilizability in aqueous
solutions of different composition are inhibited by protease inhibitors, in particular, leupeptin and aprotinin.
The presence of natural protease inhibitors in the ovarian ﬂuid that prevent spontaneous egg activation is pro-
posed. The decrease in the concentration of these inhibitors as the ovarian ﬂuid is diluted in aquatic medium
during spawning can explain egg activation in the absence of sperm.
, 12-O-tetradecanoylphorbol-13-acetate, phorbol 12,13-dibutyrate.