 |
Contenido | Volumenes anteriores | ||
CHARACTERIZATION OF SOME
TECHNIQUES OF
Microfilaments, 7-nm cytoskeletal elements, are ubiquitous in eukaryotic cells and participate in motility and changes in cell shape (Schatten et al. 1986). Cleveage is the most conspicuous change that reveals the development of an embryo, since eggs from many animals have dark cytoplams, living observations about the behavior of microtubules have been restricted and information about the microtubule system is still meager (Sawada & Schatten 1988). In general, the significance of the microtubules and microfilaments in fertilization and embryogenesis is due the spatial distribution of cytoplasmic components (ooplasmic segregation); after mitosis the chromosomes remain near the cleavage plane and form daughter nuclei just slightly past the site of the cleavage furrow. The center of the interphase aster is separated from the daughter nucleus when the egg is cleaved. The nuclei are moved to the blastomer center after cleavage is completed (Sawada & Schatten 1988). In order to explore the living cell as it changes during reproduction and development, we used immunocytochemistry and cytochernistry techniques with fluorescent and non-flurorecent labels for examining DNA, microfilament, cortical granules and microtubules (Epel 1980; Schatten 1982; Shroeder 1981; Barack et al. 1981). This work describes some aspects of fertilization and embryonic development in two small Chilean mussels (Perumytilus purpuratus and Semimytilus algosus) by means of light microscopy. MATERIALS AND METHODS Gametes from the Chilean mussels P. purpuratus and S. algosus were obtained by raising the temperature of sea water, and the fertilization was made in vitro condition. Male and female were placed at 4 °C over the night, followed by an increase in temperature to 20 °C (Loosanoff & Davis 1963). Ripe mussels of R purpuratus released large number of gametes. This method of spawning failed in S. algosus, the gametes in this specie where obtained by squashing the gonad of ripe specimens. In S. algosus autofertilization was obtained because mature ovocites and sperms of the same individual were utilized. These fertilized eggs were cultured in 1M urea pH 8,1 for at least 15 minutes, examined and if the fertilization envelopes remained the incubation was repeated; next the fertilized eggs were gently rinsed three times with calcium-free sea water containing 10 mM Tris; 2,5 mM EGTA (N, N°Tetraacetic acid), pH 8,1, until the appropriate stage was obtained. Stages of development were fixed onto poly-L-Lysine-coated coverslips (lmgr/ml polylysine) with absolute methanol during 5 minutes. Stained with 50 ug/ml rhodaminephalloidin in Phosphate Buffer Saline (PBS) (1:40) (Barak, et al. 1981; Cline and Schatten 1986) for F-Actin (Oligomeric and polymeric) for 30 minutes at room temperature, rinsed with PBS. DNA was visualized with specific compound: 4,6 diamino-2-p henylindole (DAPI) (Thombes et al. 1992); the coverslip was mounted on the top of a small drop of glycerol and examined with fluorescence microscopy epifluorescense rhodamine filters. For microtubules and DNA detection, about one hundred eggs without envelopes were gently pipeted onto polylysine coated-coverslips, treated with extraction buffer composed of 25% glycerol, 25mM Hepes, 0,5 mM MgCl, 10% Methanol, pH 6,9 with 1% Triton X-100 (Schatten et al. 1985) during 20 minutes at room temperature. The microtubules were recognized by indirect immunofluorescence (IFI) using an FITC antimouse IgG antibody (Sigma). The mouse monoclonal antibody was detected by a secondary antibody conjugated to Rhodamine and/or fluorescein, for introducing this stain into the egg without disrupting the cell surface structure, the membranes were gently permeabilized with lysolecithin (Miller et al. 1978). Dithiothreitol (DTT) was used to remove the viteline layer from unfertilized eggs (Cline and Schatten 1986). Additionally the DNA was labeled with DAPI. The cortical granules were observed in unfertilized eggs. The eggs were dejellied by several passages through 100um bolting silk, attacho monolayer by polylysine-coated slides and then their tops were sheared off by squirt of a calcium-free medium containing 10 mM DTT (Cline & Schatten 1986) and tripsine pH 8,0. The isolated eggs surfaces were examined with phase microscopy. RESULTS POLYMERIC AND OLIGOMERIC ACTIN DETECTION WITH RODAMINE-PHALLOIDIN Microfilaments were detected throughout the cortex of fertilized eggs and dividing cells of P. purpuratus and S. algous, indicating that microfilaments were found in association with the entire egg surface. In the equatorial region of dividing cells thick spots of cortical staining were detected in relationship with the leading edges of the cleavage furrow (Figure 1). Figure 1 TUBULIN DETECTION WITH ANTITUBULIN MONOCLONAL ANTIBODY Microtubules detection was made in D larval stage by first labelling with monoclonal anti-ß-tubulin antibody. The immunofluorescence was observed in the surface of the vellum and in the cilium. DNA was labeled with DAPI in the same specimens (Figure 2). Figure 2 DNA DETECTION WITH DAPI DNA was clearly visualized using DAPI in unfertilizing eggs, fertilized eggs and blastomeres of larval stages of P. purpuratus and S. algosus. In unfertilized eggs staining DNA was incorporated to the chromosomes of female nucleus of the oocyte (Figure 3) and male pronucleus of the sperm. During the fertilization process staining DNA was observed in.the nucleus of secondary oocyte and polar body (Figure 4). In larval stages DNA was observed in the nucleus of the blastomeres (Figure 2 and 5). CORTICAL GRANULES DETECTION The isolated unfertilized eggs surfaces examined with phase microscopy are seen with a uniform arrangement of cortical granule-like structures (Figure 6). Secretion was not observed when in vitro adding 1mM CaCl2 onto the isolated eggs surface. DISCUSSION The immunocytochernistry localization of microfilaments with monoclonal anti-actin antibody is very difficult by background staining due to the monomeric actin. Rhodamine-labelled phalloidin impairs this process because of detected oligomeric and polimeric actin. Rhodamin phalloidin detection of egg microfilaments was shown to provide a sensitive and easy alternative to transmission electron microscopy for studies of microfilament patterns during fertilization and early development of marine invertebrates (Bestor & Chatten 1981; Schatten et al. 1986; Sawada and Schatten 1988: Perez et al. 1991). This method permits a global detection of microfilaments in a large number of cells in different stages of development (Cline and Schatten 1986). The detection of polymerized actin in the fertilized and cleaving eggs of P purpuratus and S. algosus suggests that this microfilament plays an important role in the organization of the cytoskeleton of these mussels. During fertilization and cell division the tubulin images may delineate centrosomal structures. Centrosomes, which organize microtubule configurations, undergo replication and division process of the blastomers (Cline and Schatten 1986). The monoclonal antibody was used in D larval stages of R purpuratus and recognized by indirect immunofluorescence the presence of tubulin molecules. The detection of eggs and embryos microfilaments is shown here to provide a sensitive alternative to the complicated techniques involved in Transmission Electron Microscopy for the analysis of their changing configuration during fertilization and early development, and may well prove valuable in the investigation of other alteration in cell shape (Cline & Longo 1986). Cortical granules are specialized membrane-bound secretory granules located beneath the plasmatic membrane of most invertebrates and vertebrates eggs (Raven 1970; Guraya 1982). In most organisms that possess cortical granules, the content of the granules are discharged by exocytosis when the sperm activates the eggs during the fertilization (Schuel 1978). The exocytosis of cortical granules can be obtained by artificially increasing the concentration of Ca+ 2 (Vacquier 1975). Mussels of the genus Mytilus possess cortical granules (Humphreys 1962). The eggs of some invertebrates contain nonsecretory cortical granules that are not discharged during fertilization and persist during early embryonic development, as in the surfclam Spisula (Longo and Anderson 1970) and the mussel Mytilus (Humphreys 1967). R purpuratus may have cortical granules similar to Mytilus. A more comprehensive study is quite necessary in order to confirm his hypothesis. Ver referencias _____________________________________ 1. Embryology Institute, Biological Science Faculty, Universidad Austral de Chile, Valdivia, Chile. 2. Reproduction and Development Biology Laboratory, Biological Sciences Faculty. Universidad Nacional Mayor de San Marcos, Lima, Perú.
|