Consequences of the Spemann-Mangold organizer concept for embryological research in Russia: personal impressions.

The impact of the organizer concept on Russian experimental embryology is shortly reviewed. Attempts to study embryonic induction in Russia may be grouped into embryological and biochemical approaches. This paper provides a framework for, and overvalue of, the contributions of Russian biologists to the problem of embryonic induction. Two model systems--lens and neural inductions--are of special significance to modern developmental biologists. Moreover, the study of eye lens induction actually gave rise to research on developmental mechanics in Russia. This was one of the reasons why we limited this article to these two model systems. After retrospective consideration of the results of the search for possible lens-inducing factor candidates, the discussion turns towards some of the examples of neural-inducing agents detected in embryonic tissues and the new questions raised by the progress that has been made in the analysis of the Spemann-Mangold organizer.

Frog lim-1-like protein is expressed predominantly in the nervous tissue, gonads, and early embryos of the bivalve mollusc Mytilus galloprovincialis.

In a few well-known cases, the biological consequences of the disruption of lim-1 homeodomain (HD) genes have demonstrated the important roles of these genes in vertebrate development, especially in the nervous tissue, kidney, and gonads. Functional assay approaches require information not only about lim-1 gene organization, but also about properties and tissue localization of Lim-1 proteins. Although lim-1 genes have been identified in certain phyla of invertebrates, no information is available on Lim-1 proteins and genes in bivalve molluscs. Our study represents the beginning stage of identification of the Lim-1-related proteins in marine bivalves. Using antibodies against the C-terminal region of the Xenopus laevis Lim-1 protein, we describe cross-reactive antigen patterns in adults and early embryos of the mussel Mytilus galloprovincialis, as well as in sea urchin and chick embryos. In adult mussels, nervous ganglia and gonads display the most prominent Lim-1 immunoreactivity. Further, the antibodies verified the prediction that mussel Lim-1 antigens, like Lim-1 HD proteins in general, can be localized in the nucleus. Moreover, antibody detection allowed us to identify the Lim-1-like antigens in unfertilized mature eggs, as well as in very early embryos of bivalve molluscs and sea urchins (Strongylocentrotus purpuratus). In mussel eggs and embryos, Lim-1 antigens are expressed in multiple forms (40, 45, and 65 kDa), as detected by SDS-PAGE followed by Western blot. Taken together, the observations emphasize the conservation of the Lim-1 protein expression pattern in the nervous tissue and gonads of different animal groups, and demonstrate that Lim-1-like polypeptides can be maternally accumulated in eggs and, therefore, are present in very early embryos before zygotic expression of the genes begins.

Carboxylesterases moonlight in the male reproductive tract: a functional shift pivotal for male fertility.

This essay addresses the carboxylesterase redundancy in the male reproductive tract seemingly conserved across phyla. Evidence is provided which suggests that carboxylesterases are recruited by the male reproductive system in certain animal groups. These provide advantageous metabolic capabilities to sperm protection, sperm maturation, and sperm use. Rather than an archival record of the available data, we seek possible answers to the central question: Why is carboxylesterase over-expression adaptive with the functioning of the male reproductive tract with respect to male fertility? We discuss patterns of carboxylesterase over-expression and accumulation in different compartments of the male reproductive tract. We also provide evidence of how these patterns are associated with a long sperm path to egg through different local effects. The hyper-expression of carboxylesterases can play different physiological roles depending on its localization in the male reproductive system. However, all the "acquired" functions can serve the same purpose; creating conditions which maximize the fertilizing potential of the sperm. To confirm our concept and more clearly illuminate "moonlighting" roles of carboxylesterases in the male reproductive tract, requires a more extensive comparative analysis of a variety of carboxylesterases in a larger number of species.

Carboxylesterase overexpression in the male reproductive tract: a universal safeguarding mechanism?

Data on expression patterns of carboxylesterases in the male reproductive tract of different animal groups (i.e. bivalve mollusks, fruitflies and rodents) are summarized to highlight some particularly interesting questions in the context of sperm differentiation, maturation and function. The male reproductive system, in spite of extreme variation in the anatomical/morphological organization in different species, is characterized by similar patterns of male-dependent carboxylesterase overexpression. The phenomenon of conserved carboxylesterase overexpression indicates similar male sex-associated functions of the enzymes. There is possible evidence of carboxylesterase recruitment by male reproductive-tract tissues indicating that it could be adaptive for spermatogenesis, sperm maturation and sperm use. Moreover, this idea can be extended to include a sperm cell lineage protection. This issue is discussed in the light of recent data on environmental reproductive xenobiotics that can provide a basis for a hypothetical explanation of carboxylesterase overexpression in the male reproductive tract. Based on a well-known role of carboxylesterases in detoxification of environmental chemicals such as organophosphate pesticides, it is proposed that various male genital tract carboxylesterases may be characterized by a similar physiological function to protect the male reproductive system against xenobiotic influences that could provoke its dysfunction, thus altering sperm differentiation and maturation.

Male-associated polypeptide (MAP) expression in different compartments of the reproductive system of the mussel Mytilus galloprovincialis: immunocytochemical and western blot study.

Mytilus mussels are characterized by annually repeated reproduction which is associated with subsequent growth, morphogenesis, breakdown and redevelopment of the gonad and reproductive tract into mantle mesenchyme. We present a description of the expression of the male-associated polypeptide (MAP; see Mikhailov et al. 1995) in different compartments of the male reproductive system as well as in mantle gonad-supporting tissue. MAP is expressed in both gonad and mantle structures in dynamic patterns that show a substantial overlap in terms of dependence on the stage of gonad development/involution. In general, the total MAP concentration directly correlates with the volume of gonad tubule/duct structures but inversely correlates with mantle connective tissue cell fraction. A maximum of MAP expression is reached in the fully ripe male gonad. MAP is localized around gonad tubules/ducts, in the gonoduct epithelium, membranes of follicle-like structures as well as in the extracellular fiber-like structures of the mantle. However, we also demonstrate unique sites of MAP accumulation in the lumen of gonad follicle-like tubules and in ductal fluid. The latter is characterized by a very high MAP concentration. MAP is also detected in sperm-containing cell suspension obtained by gonad biopsy which we interpret as a result of the adsorption of MAP on mature spermatozoa. The results obtained should be taken into consideration in the interpretation of possible MAP functions since they seem to point to MAP as a major component of ductal (seminal) fluid of the male reproductive tract. It is likely that MAP is able to complement the processes of sperm terminal differentiation and maturation. In addition, we demonstrate that the male-predominant character of MAP expression is restricted by gonad-containing tissues (i.e., mantle and visceral mass) only, although the polypeptide is also detected in other somatic organs in both males and females.

Male-predominant carboxylesterase expression in the reproductive system of molluscs and insects: immunochemical and biochemical similarity between Mytilus male associated polypeptide (MAP) and Drosophila sex-specific esterase S.

We suggested that sexual differentiation of the reproductive system in gonochoric species of invertebrates can be characterized by common molecular mechanisms in spite of high morphological divergences of reproductive tract organs in different animal groups. The present study focused on this problem and report our observations on biochemical characteristics of male-associated polypeptide (MAP) identified in the gonad tissue of bivalve molluscs, Mytilus galloprovincialis, in comparison to those of male-specific carboxylesterase (esterase S) of Drosophila virilis ejaculatory bulbs. We provide evidences for the immunochemical similarity of Mytilus MAP and Drosophila esterase S. We also show that MAP is characterized by esterase activity toward both, alpha- and beta-naphthyl acetates. Using immunofluorescence, we found MAP in the gonad (mantle) connective tissue, membranes of follicles and around gonad ducts but not in sperm cells. Nevertheless, the levels of MAP expression depend on presence or absence of ripe spermatozoa in the gonad follicles. In mature gonads before spawning, MAP is expressed at high level, while in the spent gonads only traces of this polypeptide could be detected. Using Western immunoblot, MAP was not observed in spermatozoa obtained by biopsy of gonad follicles. In contrast, we found this protein in spawned sperm cells. Thus, we suggest that spawning may be required to establish the trafficking mechanisms that control whether MAP is retained or excreted by the gonad. Taken together, the results indicate that MAP of M. galloprovincialis is structurally and functionally related to esterase S of D. virilis ejaculatory bulbs.

Developmental patterns of crystallin expression during lens fiber differentiation in amphibians.

Data on activation of crystallin synthesis during lens fiber (LF) formation in amphibians are summarized to point out the questions particularly interesting in the context of lens cell lineage-specific expression programming under different developmental conditions. LFs are known to differentiate throughout life along the same pathway that includes at least five compartments. Using the amphibian eye lens as a model, we have studied how crystallins are expressed in the course of: (1) embryonic LF formation, (2) LF differentiation in adults, and (3) LF transdifferentiation from other (non-lens) eye tissues. Our experiments showed that synthesis of crystallins during morphologically similar LF differentiation in embryonic and adult amphibian lens has different spatial-temporal patterns (i.e., is apparently activated according to different programs). Certain results obtained in our studies suggest the absence of any direct relationship between the capacity of adult newt iris cells to transdifferentiate into LFs and crystallin synthesis (<> to such transdifferentiation) in them. Crystallins appear at the advanced stages of iris transdifferentiation into the lens and dynamics of their synthesis in the <> lens resembles that in the embryonic lens, although a new lens rudiment develops from the adult iris epithelium. Data on alternative patterns of the crystallin gene activation are summarized and compared with recent observations on spatial-temporal expression of Pax genes, which play an essential role in lens cell commitment and crystallin synthesis. On this basis, it is suggested that ontogenetic and tissue- or cell-specific changes in Pax gene expression may result in altered programs for activation of crystallin genes in embryonic, adult, and regenerating lens.

Sexual differentiation of reproductive tissue in bivalve molluscs: identification of male associated polypeptide in the mantle of Mytilus galloprovincialis Lmk.

We have addressed the question of sexual reproductive tissue dimorphism in bivalve molluscs, Mytilus galloprovincialis Lmk, which is a stable gonochoric species although with no apparent differences in gonad morphology of both sexes. At all periods of the annual cycle the proteins specific of male/female gonads were identified. One of these proteins, "male-associated polypeptide" with apparent MW 39 kDa (MAP-39), has been biochemically and immunochemically characterized. MAP-39 concentration in male mature gonads achieved up to 10% of the total soluble protein while in female ones only traces of this protein could be detected. In male mantle, MAP-39 expression was associated with the process of gonad development and maturation as well as gamete spawning, although this polypeptide has been localized in fibroblast-like cells, membrane of follicles and connective tissue matrix of the mantle but not in germinal cells.

Partially purified factor from embryonic chick brain can provoke neuralization of Rana temporaria and Triturus alpestris but not Xenopus laevis early gastrula ectoderm.

A high neuralizing activity has been determined in forebrain of 7.5-day old chick embryos using Rana temporaria early gastrula ectoderm as reacting tissue (Mikhailov and Gorgolyuk, Soviet Scientific Reviews, Section of Physiology and General Biology, Vol. 1: 267-306, 1987). The corresponding protease-sensitive agent was extracted, partially purified by chromatography on DEAE-Toyopearl and Heparin-Ultragel columns, and its neuralizing activity was tested in vitro on ectoderm isolated from early gastrulae of R. temporaria, Triturus alpestris, and Xenopus laevis at different concentrations and for different periods of time (animal cap assay). Induction of neural structures was found in R. temporaria and T. alpestris explants (up to 100 and 60%, respectively), but not in cultures of X. laevis ectoderm. Under our experimental conditions, so-called "autoneuralization" of the ectoderm explants can safely be excluded. The results are discussed in relation to the neural competence of amphibian ectoderm and the mechanisms of neuralizing actions of different factors which might be involved in neural induction and patterning.

[The crystalline lens as a model for studying the cellular and molecular mechanisms of differentiation in the adult organism]. / Khrustalik kak model' dlia izucheniia kletochnykh i molekuliarnykh mekhanizmov differentsirovki vo vzroslom organizme.

This review summarizes the published and authors own data about characteristics of expression of tissue-specific lens proteins (crystallins) during differentiation of lens epithelial cells into lens fibers in adult mammals, birds, reptiles, and amphibians. Information is analyzed about the presence, synthesis, and localization of different crystallins in lens epithelium and fiber cells of lens cortex and nucleus which correspond to sequential stages of lens cell differentiation. The data available suggest that morphologically similar differentiation of lens epithelial cells into fibers in different classes of vertebrates is accompanied by different programs for activation (and/or increase) of tissue-specific protein synthesis.

[The embryonic brain-derived neuralizing factor (EBDNF): its partial purification by ion-exchange and affinity chromatography]. / Neiralizuiushchii faktor iz émbrional'nogo golovnogo mozga (EBDNF): chastichnaia ochistka s pomoshch'iu ionoobmennoi i affinnoi khromatografii.

The data are presented about isolation from the brain of 7.5-day chick embryos of a factor capable of neuralizing effect on the early gastrula ectoderm of the grass frog Rana temporaria L. Earlier this factor was defined as embryonic brain-derived neuralizing factor (EBDNF) (Mikhailov, Gorgoliuk, 1989). The isolation procedure includes (1) extraction with deionized water at pH 9.0; (2) ion exchange chromatography on a column with DEAE-adsorbent at pII 8.0; (3) affinity chromatography on Heparin-Ultragel column. EBDNF-containing fraction is eluted from the Heparin-Ultragel column with 250 mM NaCl as a separate peak. Four bands are observed on SDS-electrophoregrams of this fraction, two more prominent ones having the molecular weight of 43 and 63 kDa. The yield of EBDNF-containing fraction is about 0.01-0.02% of the wet weight of the initial brain tissue.

[Forskolin and cyclic adenosine monophosphate inhibit the neurulation action of concanavalin A on explants of the gastrula ectoderm of amphibians]. / Forskolin i tsiklicheskii adenozin monofosfat ingibiruiut neitralizuiushchee deistvie konkanavalina A na ékstrakty éktodermy gastruly amfibii.

We studied effects of forskolin, an activator of adenylate cyclase activity, and dioctanoyl-cyclic adenosine monophosphate (do-cAMP) on neutralizing (N) activity of concanavalin A (Con A). Biological testing was performed using explanted animal pole ectoderm of the Rana temporaria early gastrula. Con A treatment (200 micrograms/ml, 2 h) resulted in neutralization of 70-90% explants. If the explants were previously treated with forskolin (100 microM, 1 h), Con A effect decreased to 10%. When Con A and forskolin were applied simultaneously, no N-effect was observed. The same results were obtained with simultaneous treatment of the explants with Con A and do-cAMP (10(-5) M). Moreover, treatment with forskolin of the explants previously treated with Con A inhibited their neural differentiation. We suggest that N-effect of Con A is calcium dependent; the increase in intracellular cAMP after treatment of explants with forskolin or do-cAMP interferes with intracellular Ca2+ release and this results in the inhibited N-effect of Con A.

[Plant lectins and embryonic tissue factors as probes for studying the mechanisms of neural induction in amphibians]. / Rastitel'nye lektiny i faktory iz émbrional'nykh tkanei kak zondy dlia issledovaniia mekhanizmov neiral'noi induktsii u amfibii.

Using various experimental techniques, we have demonstrated that animal pole ectoderm (APE) of Rana temporaria embryos at the stage of early gastrula is a good target tissue for testing the neuralizing (N) factors. In this respect R. temporaria APE is comparable with APE of some other amphibian species. We found that concanavalin A (con A), phytohemagglutinin (PHA) and embryonic brain-derived neuralizing factor (EBDNF; a factor extracted from the chick embryonic brain and partially purified) have a pronounced N-effect on the APE of R. temporaria. In order to analyse possible mechanisms of N-action of these factors, we have cultured APE explants for 3 or 18 h in the medium containing various concentrations of con A, PHA of EBDNF. All these factors could produce neuralization in 50% explants. However, the optimal concentration and time of exposure were different. This is an evidence for different mechanisms of reception and transmission of a N-signal in each particular case. It appears that the APE consists of several cell subpopulations which differ in their threshold sensitivity to the N-effect of studied agents.

[The synthesis and localization of crystallins in different cell compartments of the crystalline lens in adult frogs: immunoautoradiographic and immunofluorescent research]. / Sintez i lokalizatsiia kristallinov v razlichnykh kletochnykh kompartmentakh khrustalika glaza vzroslykh liagushek: immunoavtoradiograficheskoe i immunofluorestsentnoe issledovanie.

The purpose of this study was to analyze immunochemically the synthesis and distribution of tissue-specific proteins, i.e., alpha-, beta- gamma- and rho-crystallins, in morphologically distinct regions of the frog (Rana temporaria L.) lens which consist of cells at various stages of differentiation, maturation and aging. Five such cell compartments can be distinguished in the lens: (1) central zone of lens epithelium (stem/clonogenic cells); (2) equatorial epithelial cells (differentiating cells); (3) lens fibers of the outer cortex (post-mitotic differentiated cells); (4) lens fibers of the deep cortex (cells without nuclei at terminal stage of differentiation); and (5) cells of the lens "nucleus" (cells formed during embryogenesis). Intact lenses and isolated lens epithelium were cultured in vitro in the presence of 35S-methionine. Then lens epithelium, outer and deep cortex, and lens nucleus were extracted with buffered saline and extracts used for immunoautoradiography. Distribution of crystallins in paraffin sections of the whole lens or isolated lens epithelium was studied using indirect immunofluorescence. Synthesis of alpha-crystallins was observed in lens epithelium and cortex, but not in lens nucleus. According to immunohistochemistry, these proteins were absent from central part of the lens epithelium: positive fluorescence was observed only in elongating cells at its periphery and in lens fibers. The data on beta-crystallins are similar except that synthesis of these proteins (traces) was detected also in lens nucleus. Synthesis of gamma-crystallins was detected in lens cortex and nucleus (traces) but not in epithelium. Immunohistochemistry showed that these proteins are absent from all regions of lens epithelium and found only in fiber cells of cortex and nucleus. Rho-crystallin was synthesized in all cell compartments of the adult lens, and all lens cells contained this protein. Our results show that cells of central lens epithelium do not contain alpha- beta- or gamma-crystallins (or the rate of their synthesis is insignificant). While cells are moving towards lens equator and elongating, synthesis of alpha- and beta-crystallins is activated. Gamma-crystallins are synthesized later, first in young lens fibers near lens equator. During embryonic development in amphibia, in contrast, gamma- and beta-crystallins are detected at earlier stages than alpha- and rho-crystallins (Mikhailov et al., 1988). These data suggest that different mechanisms are involved in differentiation on lens fibers from embryonic precursor cells, on one hand, and from epithelial stem cells of adult lens, on the other.

[Concanavalin-binding proteins and cytokeratins in different tissues of the early amphibian gastrula (Rana temporaria, Xenopus laevis)]. / Konkanavalin-sviazyvaiushchie belki i tsitokeratiny raznykh tkanei rannei gastruly amfibii (Rana temporaria, Xenopus laevis).

Concanavalin A (con A), a lectin which specifically interacts with aD-mannose and aD-glucose, has a neutralizing effect on the explants of the early gastrula ectoderm of several amphibian species. Consequently, it was interesting to study con A-binding protein spectrum of the ectoderm and compare it to those of other early gastrula tissues. Animal pole ectoderm (APE), dorsal blastopore lip (DBL) and vegetal pole endoderm (VPE) were dissected from early gastrulae of Rana temporaria and Xenopus laevis. The extracts were subjected to SDS-PAGE with subsequent immunoelectroblotting on nitrocellulose membranes. The blots were sequentially treated with con A solution, horseradish peroxidase and diaminobenzidine. Spectra of the con A-binding glycoproteins were similar in APE, DBL and VPE of R. temporaria. Ten-twelve fractions with the molecular weight in the range from 30 to 150 kDa were stained in each blot. Fractions with the molecular weight of 150, 125, 104, 94 and 42 kDa showed more prominent lectin binding. Con A-binding protein spectra remained unchanged after freezing-thawing of the studied extracts, as well as after blots were treated with neuraminidase or sulphuric acid in order to remove sialic acid residues; the only exception was 42 kDa fraction. At the same time, a-methyl-D-mannoside pyranoside completely blocked con A binding by fractions of the studied extracts. In histological sections of R. temporaria early gastrula, all cells bound FITC-labelled con A. Similar data were obtained with tissues of X. laevis early gastrula. While electrophoretic pattern of X. laevis tissues drastically differed from that of R. temporaria, there were no significant differences between con A-binding protein spectra of X. laevis APE, DBL or VPE. Thus, all studied tissues of the amphibian early gastrula contain similar set of con A-binding proteins; however, only APE is capable of neutralization in response to con A action. These data favor our earlier assumption (see Mikhailov et al., 1989) that con A reception and transmission of the corresponding signal do not determine the characteristics of the target cells response. APE, DBL and VPE extracts were assayed also for the presence of a protein similar to cytokeratin No. 8 characteristic of simple epithelia of mammals. Experiments were performed using immunoelectroblotting with monoclonal antibodies (mAB) against cytokeratin No. 8 from rat colon (mAB E2 and E7 kindly supplied by Dr. G. A. Bannikov). In R. temporaria embryos, cytokeratin 8 was detected in APE, but not in DBL or VPE. In X. laevis gastrulae all the tissues studied contained this cytokeratin.