[Fish ovarian fluid contains protease inhibitors].

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 particular, type I serpin a, a protein inhibitor of trypsin proteases.

[Spontaneous activation of fish eggs is abolished by protease inhibitors].

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 fertilization. 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 proposed. 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.

[A study of proteins of annexin group in early fish development. IV. Identification of calcium-binding proteins in zebrafish egg by mass spectrometry].

Calcium-binding proteins were isolated from zebrafish (Brachidanio rerio) eggs by the method of precipitation with acidic phospholipids in the presence of calcium ions. The revealed proteins separated by SDS-PAGE were identified by mass spectrometric tryptic peptide fingerprinting. The proteins included annexins A2a, A1a, A13.1, and A5. In addition, copine III, a member of the recently described copine family of C2 domain-containing proteins, was identified. Total RNA was analyzed in mature oocytes by RT-PCR and transcripts of two different annexin A13 forms (A13.1 and A13.2) as well as of annexin A3 were detected. Thus, the presence of both proteins and mRNAs of annexins has been shown in the zebrafish egg.

[A study of annexin family protein in early development of fish. II. Analysis of complete amino acid sequence]. / Izuchenie belka gruppy anneksinov v rannem razvitii ryb. II. Opredelenie polnoi aminokislotnoi posledovatel'nosti.

We studied mRNA structure of 31 kDa annexin of zebra fish Brachydanio rerio using previously obtained 3'-terminal incomplete cDNA. The size of this protein mRNA was determined by Northern hybridization. PCR screening of cDNA library of zebra fish gastrula allowed us to obtain cDNA of the 5'-terminal regions of the mRNA. The primary structure of the protein deduced from the mRNA sequence allowed us to identify it as an annexin IV with threonine in position 6--a phosphorylation target for protein kinase C.

[The temperature dependence of selection efficiency and the expression of the genes controlling the rate of embryonic development in Drosophila melanogaster]. / Temperaturnaia zavisimost' éffektivnosti selektsii i ékspressii genov, kontroliruiushchikh skorost' émbrional'nogo razvitiia u Drosophila melanogaster.

We have already shown that selection of heterogeneous D. melanogaster populations for the rate of embryogenesis at 32 degrees C may produce populations, in which 50% of the larvae are hatched 20-40 min earlier, i.e., 2-4% more rapidly than, in the control. Highly inbred strains were obtained from the selected populations and develop 5-7% more rapidly than the control populations. Here we studied separately the effects of temperature on the efficiency of selection and expression of the selected genotype. With this in view, multiple (9-15 rounds) selection of the first 10% of the larvae was performed at 17, 25, and 32 degrees C and the mean duration of embryogenesis (hatching of 50% larvae) at these three temperatures was determined in the entire selected population. Selection proved to be almost equally efficient at all temperatures. On the contrary, expression of the selected genotypes markedly depended on temperature: the rate of embryogenesis of the selected populations exceeded that in the control at 17 degrees C by 1.1%, at 25 degrees C by 2.5%, and at 32 degrees C by 3.5%. In the inbred strains this dependence was even more pronounced: 1.2%, 2.7%, and 5.7%, respectively. The temperature dependence of expression of the genes collected by selection and coding for accelerated development means that these genes affect the rate of development as the function of temperature by changing the angle of the curve inclination. Possible mechanisms of this phenomenon are discussed.

[Properties of inbred Drosophila melanogaster lines obtained from a population selected for an increased rate of embryonic development]. / Svoistva inbrednykh linii Drosophila melanogaster, poluchennykh iz populiatsii, selektirovannoi na povyshennuiu skorost' émbrional'nogo razvitiia.

Two heterogeneous Drosophila melanogaster populations were subjected to selection for an increased rate of embryonic development by picking out the first 10% of hatching larvae. After repeating this procedure in 15 generations, "fast" populations were obtained, in which the duration of embryonic development at high temperature (31-32 degrees C) was 30-40 min less than in nonselected control populations. The results of preliminary experiments on substituting the second and third chromosomes in the selected and control populations provide evidence that selected genes responsible for accelerated development are located on the second chromosome. Inbreeding in 12 generations of selected populations was used to obtain about 40 lines homozygous, in particular, at the alcohol dehydrogenase gene. In four lines, the developmental rate was higher than in a homozygous control line, but others did not differ from control or developed more slowly. The duration of embryonic development at 32 degrees C in fast lines was 50-70 min shorter than in control, but this difference was significantly less at lower temperatures (25 and 17 degrees C). Hence, high temperature is primarily a factor in providing conditions for the expression of genes determining the developmental rate, rather than a factor of selection for these genes. It is suggested that selected genes modify developmental rate dependence on temperature.

Phalloidin inhibits cortical granule exocytosis and ooplasmic segregation in loach eggs.

Injections of phalloidin under the surface of loach eggs, followed by activation of the eggs in tap water, result in local inhibition of cortical granule (CG) exocytosis. Light and electron microscopy revealed that in the region where exocytosis is inhibited the thickness of the microfilamentous cortex (MC) separating CGs from the plasma membrane (PM) is increased significantly, and many CGs are detached and have moved away from the MC. Injections of phalloidin also inhibit ooplasmic segregation in fertilized eggs. The experiments suggest that in intact eggs the MC represents a physical barrier to CG exocytosis, and that interactions of the MC with the PM and CGs are crucial for the retention of CGs near the sites of fusion.