Fish ovarian fluid contains protease inhibitorsby A. A. Minin, S. G. Ozerova

Russ J Dev Biol


Developmental Biology


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ISSN 10623604, Russian Journal of Developmental Biology, 2015, Vol. 46, No. 1, pp. 33–37. © Pleiades Publishing, Inc., 2015.

Original Russian Text © A.A. Minin, S.G. Ozerova, 2015, published in Ontogenez, 2015, Vol. 46, No. 1, pp. 38–43. 33


In sexual reproduction of animals, activation (trig gering the zygote development program) usually occurs as a result of interaction between the egg and the sperm. One of the earliest events during activation is the formation of the socalled fertilization mem brane as a result of exocytosis of cortical granules, which, in most groups of animals, is triggered by the penetration of the sperm into the egg and serves to pre vent polyspermy. However, the process of egg activa tion in fish has some unique features.

It is known that, in the spawning medium, eggs of the majority of fish retain the ability for fertilization for a limited time characteristic of each species due to the socalled spontaneous (i.e., without sperm) activa tion, which is characteristic for this group of verte brates. The conditions under which the eggs of differ ent species of fish are activated and fertilized were described and considered in detail in the monograph by A.S. Ginzburg (1968). Similarly to normal activa tion, spontaneous activation is accompanied by the formation of the fertilization membrane, preventing the penetration of sperms into the egg. A characteristic feature of fish is external fertilization; therefore, the process of spontaneous activation, which is triggered by the contact of eggs with the spawning environment, determines the time of possible fertilization. It should be noted that, during the maturation and ovulation, eggs can retain for some time the ability for fertiliza tion in the gonads of fish before spawning, being located in a specific environment formed by the con tents of the ruptured follicles and tissue fluid in the gonad cavity. A.S. Ginzburg proposed the term “coelomic fluid” for this medium.

The spontaneous (i.e., occurring without sperm) activation, which is observed when fish eggs get into the spawning medium, generally does not lead to the development of a haploid embryo but is accompanied by the formation of the fertilization membrane, abnormal ooplasmic segregation, incorrect cleavage, and, eventually, the death of the unfertilized eggs.

The activation of the egg is accompanied by an increase in the concentration of free calcium ions in the cytoplasm (Gilkey et al., 1978, Lee et al., 1999).

The exocytosis of cortical granules in the fish egg, resulting in the formation of the fertilization mem brane, is probably activated by the calcium ions and involves a series of events, including the cortical actin reorganization in the egg (Ivanenkov et al., 1990,

Becker and Hart, 1999). It was shown that the sponta neous activation of fish eggs, similarly to the normal fertilization, is accompanied by a wavelike increase in the concentration of calcium ions in the cytoplasm and exocytosis of cortical granules (Lee et al., 1999); however, the signaling mechanisms triggering this increase are obscure. Thus, it can be assumed that the spontaneous activation as a general phenomenon characteristic of fish eggs is closely related to the pro cesses of reproduction in fish and plays a key role in determining the temporal parameters of fertilization.

The authors of some studies, who investigated the activation of fish eggs without sperm by incubating them in salt solutions of different composition (Hart and Yu, 1980; Lee et al., 1999, Gilkey et al., 1999), showed that some salt media slowed down the sponta neous activation and that only the ovarian (coelomic) fluid allows fish eggs to retain the ability for fertiliza tion for a long time (CorleySmith et al., 1995; Minin and Ozerova, 2008a). It is not necessary to take the ovarian fluid from the fish of the same species; in par ticular, in studies performed with the zebrafish eggs, salmon ovarian fluid is often used (CorleySmith et al., 1995).

Fish Ovarian Fluid Contains Protease Inhibitors

A. A. Minin and S. G. Ozerova

Koltzov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26, Moscow, 119334 Russia email:

Received August 13, 2014; in final form, September 5, 2014

Abstract—Studies of the conditions under which fish egg is activated spontaneously without the sperm showed that the egg retains the ability for fertilization in the ovarian (coelomic) fluid, which surrounds it in the gonad cavity after ovulation. Earlier, we showed that, in artificial media, the spontaneous activation is suppressed by protease inhibitors. In this study, we investigated the presence of natural protease inhibitors in the ovarian fluid and showed that the ovarian fluid of zebrafish and loach contains protease inhibitors, in par ticular, type I serpin a, a protein inhibitor of trypsin proteases.

Keywords: egg activation, protease inhibitors, Misgurnus fossilis, Brahidanio rerio, serpina1

DOI: 10.1134/S1062360415010063




We have raised the question as to which compo nents of ovarian fluid prevent the activation of fish eggs and what is the mechanism underlying the activating effect of the spawning environment. It was found that salt media containing the inhibitors of proteolytic enzymes (proteases) prevent the spontaneous activa tion of fish eggs and maintain their ability for normal fertilization (Minin and Ozerova, 2008a). It is also known that mature zebrafish follicles contain mRNA and protein of the trypsin protease inhibitor serpin a1 (KnollGellidda et al., 2006). We hypothesized that the protection of eggs from spontaneous activation attempted to answer the question as to whether the ovarian fluid of fish contains protease inhibitors.


In this study, we used the ovarian (coelomic) fluid of three cyprinid species—loach (Misgurnus fossilis), carp (Cyprinus carpio), and zebrafish (Brahidanio rerio).