Egg Of Discoglossus Pictus Research Articles

Spectrin and Ankyrin-like Proteins in the Egg of Discoglossus pictus (Anura): Their Identification and Localization in the Site of Sperm Entrance versus the Rest of the Egg: (spectrin/ankyrin/anuran eggs/fertilization).

The Discoglossus pictus egg has a specific site of sperm-egg interaction, the dimple, which has a well-defined cytoskeleton. We studied whether there are cytoskeletal and cytoskeleton-related proteins typically involved in the polarization of plasma membrane proteins. The identity and the localization of the molecules cross-reacting with antispectrin, antifodrin and antiankyrin antiobodies were investigated by immunofluoresecence and immunoblotting of the proteins of the dimple (D) and of the rest of the egg (dimple-less-egg; DLE). Two polypeptides of about 254-and 246-kD were detected in the D and DLE, and localized in the egg cortex. A third molecule, weakly cross-reacting with antispectrin and antifodrin, was found in the subcortical cytoplasm. The 246-kD polypeptide was labile in samples prepared for SDS-PAGE; a mild prefixation of eggs prevented its dispersion. Mild fixation was also needed to retain antispectrin reactivity in cryostat sections of the DLE cortex, while this is not necessary in D. A molecule of about 204-kD, cross-reacting with antiankyrin, was detected in the cortex of the whole egg. These data and the finding that the concentrations of both the 254-kD polypeptide and ankyrin are about 12-fold higher in D than in the DLE, suggest that, in D, spectrin has a specific organization.

Cytochemical Ca2+ Distribution in Activated Discoglossus pictus Eggs: A Gradient in the Predetermined Site of Fertilization.

The K-pyroantimonate/OsO4 (PA) cytochemical method coupled with EGTA and X-ray microanalytical controls has been used to localize Ca2+ at egg activation in Discoglossus pictus eggs. The results show that: 1) the PA method is able to selectively localize Ca2+ pools mobilized by activating stimuli; 2) the smooth endoplasmic reticulum (SER) elements located in the animal dimple region, i.e. in the predetermined site of fertilization, are the first egg components labeled by precipitates; 3) a decreasing gradient of precipitates is present from the center beyond the boundaries of the dimple region; 4) precipitates are lacking in the remainder of the egg even at late times after activation. The possibilities are discussed that a) SER is the major Ca2+ -releasing store at activation in Discoglossus, and b) the observed gradient of pyroantimonate-detected Ca2+ reflects an ionic Ca2+ gradient.

Antispectrin antibodies stain the oocyte nucleus and the site of fertilization channels in the egg of Discoglossus pictus (Anura)

In Discoglossus pictus eggs, only the dimple contains ionic channels active at fertilization; in particular, chloride channels are found in the central portion of the dimple, which is also the site of sperm penetration. Moreover the dimple hosts an imposing cytoskeleton, consisting of a cortical network and bundles of microfilaments extending from the microvilli. Since spectrin cross links actin and is connected through ankyrin to anion transporters in the plasma membrane of erythrocytes as well as to anion channels in other cells, we studied, in D. pictus egg, the relationship between the localization of spectrin and the high polarization of ionic channels and cytoskeletal organization. By means of immunocytochemistry, we localized spectrin exclusively in the egg dimple. In an attempt to trace back the source of spectrin localization, we immunostained sections of D. pictus ovary and localized spectrin in the nuclei of previtellogenic oocytes, where actin is also present. Antispectrin staining remained until germinal vesicle breakdown. By contrast, a cortical localization was found only when the oocytes divided into two hemispheres and into the germinative area (GA), which, after germinal vesicle breakdown, gives rise to the dimple. At this stage the antispectrin signal was particularly strong in the GA. Using Rho-pialloidin, we also established that spectrin is generally present where F-actin is found. However, spectrin and F-actin do not have the same pattern of fluorescence. In conclusion, our data suggest that spectrin may play a role in oocyte and egg polarity. In eggs, it could be instrumental in anchoring to the cytoskeleton membrane proteins such as receptors and ionic channels, including chloride-permeable channels.

The cortical reaction in the egg of Discoglossus pictus: A study of the changes in the endoplasmic reticulum at activation

In Discoglossus pictus previous ultrastructural observations have shown that at the animal dimple, where sperm fuse with the egg, cortical granules (CG), vacuoles, and tightly packed clusters of small cisternae are present. At fertilization the clusters open (i.e., become loose) and give rise to longer cisternae arranged in whorls and chains which migrate toward the plasma membrane. The vacuoles fuse to form cisternae and exocytose along with the CG. In the rest of the egg periphery, while exocytosis occurs, the clusters do not open as a result of activation ( C. Campanella, R. Talevi, U. Atripaldi, and L. Quaglia (1986). In “Molecular and Cellular Biology of Fertilization” (J. L. Hedrick, Ed.). Plenum, New York ). We have recently conducted electrophysiological studies which have detected inward currents at the dimple center, outward current at the rest of the egg surface, and an eightfold increase in [Ca 2+] i which propagates from the site of activation throughout the egg ( R. Nuccitelli, D. Kline, W. Busa, R. Talevi, and C. Campanella (1988). Dev. Biol. 130, 120–132 ). In this paper we have asked whether the anionic current and the Ca 2+ increase could be causally related to the changes of the smooth endoplasmic reticulum (SER) at activation. The results obtained by activating the eggs in ion-substituted Ringers indicate that (1) the migration of cisternae is not dependent on the polarity of the activation current crossing the dimple, but is strongly impaired, together with CG exocytosis, by 5× Cl − Ringer; (2) TMB-8, a drug which partially blocks calcium release ( C. Y. Choiu and M. J. Malagodi (1975). Brit. J. Pharmacol. 53, 279–288 ), partially inhibits opening of cisternae clusters and the formation of an SER network in the dimple. This suggests a causal relationship between the Ca 2+ rise and the cluster transformation at activation.

The cortical endoplasmic reticulum and its possible role in activation of Discoglossus pictis (Anura) eggs.

The role of endoplasmic reticulum was investigated in the egg of Discoglossus pictus; recent findings suggest that this organelle is the source of Ca2+ sequestration and release at activation. In the egg of Discoglossus the dimple is the only site where sperm-egg fusion occurs. Microvilli containing microfilament bundles penetrate into the dimple cytoplasm and thus define a cortical layer containing cortical granules, tubular cisternae 35 nm thick and vacuoles. In the underlying cytoplasm are clusters of small cisternae and mitochondria. In the region of the egg cortex outside of the dimple, clusters of cisternae, some vacuoles and a heterogeneous population of small granules are found. In eggs activated by pricking contraction starts from the site of pricking and travels to the antipode. Sections of eggs, fixed 20 to 60 s following fertilization or pricking, show that the tubular cisternae have disappeared and the clusters of cisternae have opened to give rise to longer cisternae arranged in chains. These chains pile up below the vacuoles which are now flat and long because of multiple fusion. A network of cisternae is thus formed whose constituents lean against the microfilament bundles and within 5 min from activation approach the plasma membrane. The flattened vacuoles and the cortical granules are exocytozed. Some cisternae fuse with the microvillar membrane thus participating in microvilli elongation. The microfilament rootlets grandually rearrange and become shorter. In the cortex outside the dimple the cisternae clusters do not open, whereas exocytosis of granules occurs. In eggs treated with A23187, vacuoles and cortical granules undergo exocytosis; however a cisternal network is not formed. The possible stimuli that cause the cisternae network to form at activation only in the dimple and in a matter of seconds are discussed. Our observations, namely exocytosis, formation of cortical wave of contraction and changes in the cytoskeleton organization at activation are consistent with an increase of free Ca2+. Vacuoles and cisternae appear to be good candidates for the sequestration and release of Ca2+ as well an important source for the increase in total plasma membrane at activation.

Ultrastructural and lectin binding changes during the formation of the animal dimple in oocytes of Discoglossus pictus (Anura)

In Discoglossus pictus eggs, the animal dimple is the only place where fertilization can occur. The carbohydrate composition of the dimple membrane was investigated using fluorescent-labeled lectins. Receptors for soybean agglutinin (SBA) and wheat germ agglutinin (WGA) are specifically localized on the dimple walls, whereas fucose-like residues are only present at the bottom of the dimple. This lectin binding pattern is transient since it only appears when the dimple is completely formed and disappears immediately after fertilization. The formation of the dimple requires an increase in surface. Our ultrastructural studies suggest that membrane growth occurs through the insertion of vesicles into the plasma membrane. We hypothesize that the lectin-binding glycocomponents that we observed are inserted along with the vesicles which fuse into the membrane. These vesicles most probably originate from the annulate lamellae and cisternal stacks present in full-grown oocytes. The possibility that the surface features of the dimple, such as the microvilli, the complex glycocalyx, and particulary the neutral carbohydrate residues (fucose-like), might favor sperm-egg fusion is discussed.

The Site of Spermatozoon Entrance in the Unfertilized Egg of Discoglossus pictus (Anura): An Electron Microscope Study

The egg of Discoglossus pictus has an animal dimple, a pit located at the animal hemisphere pole, which is the only site successfully penetrated by the sperm. Ultrastructural analysis of the unfertilized egg surface reveals unique features characterizing the animal dimple. They are: I) regularly spaced, finger-like microvilli supported by bundles of fibers which appear to be microtubules and surrounded by antennular glycocalyx, present at the surface of the animal dimple. The orientation and distribution of the fibers suggest that they may play an essential role in maintenance of the shape of the animal dimple: 2) granules present in the peripheral cytoplasm of the animal dimple whose characteristics are somewhat different from those of the typical Anuran cortical granules and which are absent from the rest of the peripheral cytoplasm of the egg. The unique presence of these granules at the animal dimple and their apparent loss at the time of fertilization suggest that they might actively participate in the fertilization process. Freshly laid eggs of Discoglossus picius (painted frog) exhibit a depression (the animal depression) at the animal hemisphere whose center part is invaginated and forms the animal dimple. The animal depression is filled with a gelatinous lens-shaped material forming the animal plug, which protrudes at the surface of the egg (Hibbard, 1928). The eggs are further covered with four gelatinous envelopes with different chemical compositions. The innermost envelope is made up of two membranous layers E1 and E2 and strictly adheres to the egg surface, except over the animal dimple where they are separated from the egg surface by a substance of glycoprotein nature (Ghiara, 1960). The nucleus, arrested in metaphase of the 2nd meiotic division, is located in the cortical cytoplasm of the area germinativa which is the bottom of the animal dimple. In sections of eggs at the level of the animal dimple, and particularly at its basal portion, one can clearly distinguish striations which are perpendicular to the egg surface (Hibbard,

Regional Morphological and Cytochemical Differentiation in the Fertilized Egg of Discoglossus pictus (Anura)

A vertical column of cytoplasm poor in yolk (CPY) is located in the centre of the animal region of the unfertilised and fertilised egg of Discoglossus pictus. At the base of this column is found a central region of CPY designated as “clear cytoplasm”. Cytochemical methods show that the CPY in this whole region is rich in glycogen and RNA. By 60 min post opposition (p.o.) the pigmented cortical layer starts moving towards the future ventral side. It attains its definitive position by 90 min p.o. when the grey crescent, visible from 75 min p.o. onwards, achieves its maximal extension on the future dorsal side. The cytoplasmic column is now tilted towards the future ventral side. It progressively loses its direct contact with the cell membrane and disappears. From 90 min p.o. onwards, the “clear cytoplasm” is found progressively closer to the dorsal grey crescent cortex. When the first cleavage furrow appears at 135 min p.o., the “clear cytoplasm” is situated very near the dorsal cortex and even extends somewhat below the equator. In places a relatively thin layer of cytoplasm containing medium-sized and a few large yolk granules intervenes between the grey crescent cortex and the “clear cytoplasm”. These displacements suggest that sperm entry evokes a dorsally directed cytoplasmic movement in the animal half of the egg which, among other things, may facilitate an interaction between the vegetative yolk and the grey crescent cortex, or may directly influence the dorso-ventral polarisation of the vegetative yolk.

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In Discoglossus pictus eggs, the animal dimple is the only place where fertilization can occur. The carbohydrate composition of the dimple membrane was investigated using fluorescent-labeled lectins. Receptors for soybean agglutinin (SBA) and wheat germ agglutinin (WGA) are specifically localized on the dimple walls, whereas fucose-like residues are only present at the bottom of the dimple. This lectin binding pattern is transient since it only appears when the dimple is completely formed and disappears immediately after fertilization. The formation of the dimple requires an increase in surface. Our ultrastructural studies suggest that membrane growth occurs through the insertion of vesicles into the plasma membrane. We hypothesize that the lectin-binding glycocomponents that we observed are inserted along with the vesicles which fuse into the membrane. These vesicles most probably originate from the annulate lamellae and cisternal stacks present in full-grown oocytes. The possibility that the surface features of the dimple, such as the microvilli, the complex glycocalyx, and particulary the neutral carbohydrate residues (fucose-like), might favor sperm-egg fusion is discussed.