Chiara Campanella

Ultrastructural observations on cortical endoplasmic reticulum and on residual cortical granules in the egg of Xenopus laevis

Two kinds of cortical granules (CG) are present in the unfertilized egg of Xenopus laevis: CGa and CGv. The cisternae surrounding both CGs have been found to be interconnected through elements of ER. CG cisternae also anastomose with the subcortical ER. It is suggested that these connections establish a functional unity between the CGs and that the cortical network may be involved in the propagation of the activation stimulus leading to the gradual CG activation and rupture. About 2% of the CGs are left in the cortex following activation. They have never been found in the process of extrusion in eggs fixed later than 5 min after pricking. Notable ER modifications occur in the egg cortical cytoplasm within the first 30 min following activation. Residual CGs do not show preferential distribution with regard to their position in the cortical cytoplasm or with regard to their presence in the animal and vegetal hemispheres. Most CGv and a few CGa undergo changes, although the changes are different in the two classes of granules; both CG types lose most of their original contents. CGv eventually disaggregate, whereas CGa show a reduction of their contents and form one or more large invaginations. Autophagosomes are found in the animal hemisphere 20 to 30 min after activation. They are located in the cortical cytoplasm and have a granular content similar to modified CGs.

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

Abstract 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.

Phosphorylation of nm23-H1 by CKI induces its complex formation with h-prune and promotes cell motility

The combination of an increase in the cAMP-phosphodiesterase activity of h-prune and its interaction with nm23-H1 have been shown to be key steps in the induction of cellular motility in breast cancer cells. Here we present the molecular mechanisms of this interaction. The region of the nm23–h-prune interaction lies between S120 and S125 of nm23, where missense mutants show impaired binding; this region has been highly conserved throughout evolution, and can undergo serine phosphorylation by casein kinase I. Thus, the casein kinase I δ-ɛ specific inhibitor IC261 impairs the formation of the nm23–h-prune complex, which translates ‘in vitro’ into inhibition of cellular motility in a breast cancer cellular model. A competitive permeable peptide containing the region for phosphorylation by casein kinase I impairs cellular motility to the same extent as IC261. The identification of these two modes of inhibition of formation of the nm23-H1–h-prune protein complex pave the way toward new challenges, including translational studies using IC261 or this competitive peptide ‘in vivo’ to inhibit cellular motility induced by nm23-H1–h-prune complex formation during progression of breast cancer.

Fertilization and activation potentials in Discoglossus pictus (Anura) eggs: A delayed response to activation by pricking

Abstract We have studied the electrical and morphological changes in the egg of the anuran Discoglossus pictus during fertilization and activation by pricking. Despite its atypical structure the fertilization potential (FP) in this egg resembles that of other anurans, consisting of a rapid, Cl− dependent, depolarization of some 40 mV, accompanied by a 10- to 100-fold increase in conductance. Pricking eggs at the predetermined site of sperm entry, the dimple, causes an immediate depolarization, similar to the FP, and a wave of contraction can be seen to spread from the dimple to the antipode. When eggs are pricked outside the dimple a contraction wave spreads from the puncture site to the antipode, however, the activation potential (AP) is not generated until the wave reaches the dimple. Thus the farther the puncture site from the dimple the longer the delay in the generation of the AP. Our results suggest that the contraction wave reflects a propagating Ca2+ wave and the channels activated during the AP are localized within the dimple. As there is no obvious ultrastructural change in the dimple region until several seconds after the generation of the AP it is probable that the ion channels preexist in the egg plasma membrane and are not inserted by membrane fusion.

Sperm-egg interaction in the painted frog (Discoglossus pictus): An ultrastructural study

The ultrastructure of sperm changes and penetration in the egg was studied in the anuran Discoglossus pictus, whose sperm have an acrosome cap with a typical tip, the apical rod. The first stage of the sperm apical rod and acrosome reaction (AR) consists in vesiculation between the plasma membrane and the outer acrosome membrane. The two components of the acrosome cap are released in sequence. The innermost component (component B) is dispersed first. The next acrosome change is the dispersal of the outermost acrosome content (component A). At 30 sec postinsemination, when the loss of component B is first observed, holes are seen in the innermost jelly coat (J1), surrounding the penetrating sperm. Therefore, this acrosome constituent might be related to penetration through the innermost egg investments. At 1 min postinsemination, during sperm penetration into the egg, a halo of finely granular material is observed around the inner acrosome membrane of the spermatozoon, suggesting a role for component A at this stage of penetration.

p27(BBP)/eIF6 acts as an anti-apoptotic factor upstream of Bcl-2 during Xenopus laevis development

p27BBP/eIF6 (β4-binding protein/eukaryotic initiation factor 6) regulates the joining of 40S and 60S ribosomal subunits, on receptor for activated C kinase 1 binding and protein kinase C phosphorylation in serine 235. In Xenopus, p27BBP/eIF6 is abundantly expressed in the majority of the embryonic anlagen. Although p27BBP/eIF6 abundance may be required for a general regulation of protein synthesis, our data suggest that p27BBP/eIF6 may target the translation of specific mRNAs. We injected Xp27BBP/eIF6 mRNA in one blastomere of two-cell-stage embryos and obtained a bent phenotype, the curvature being lateral with respect to the embryo antero-posterior axis. The injected side had fewer apoptotic cells than the uninjected side, whereas cell proliferation appeared unaffected. Accordingly, in Xp27BBP/eIF6 morphants, endogenous apoptosis increased. Injection of Xp27BBP/eIF6 point mutants indicated that the anti-apoptotic action of Xp27BBP/eIF6 requires the conserved S235. The bent phenotype was also obtained with B-cell lymphoma gene-2 (Bcl-2) overexpression and was rescued by Bcl-2-associated X protein (Bax)/Xp27BBP/eIF6 co-injection. In addition, embryos overexpressing Xp27BBP/eIF6 had a higher amount of Bcl-2 and an unchanged amount of Bax with respect to controls. In Xp27BBP/eIF6 morphants, Bcl-2 levels were unaffected and Bax levels were higher than in the controls. Thus, we propose that Xp27BBP/eIF6 is part of a mechanism acting on the specific translation of messengers regulating cell survival. In particular, we suggest that Xp27BBP/eIF6 may regulate the translation of factors upstream of Bcl-2/Bax.

Multiple-Particle-Tracking to investigate viscoelastic properties in living cells

Cell mechanical properties play an important role in determining many cellular activities. Passive microrheology techniques, such as Multiple-Particle-Tracking (MPT) give an insight into the structural rearrangements and viscoelastic response of a wide range of materials, in particular soft materials and complex fluids like cell cytoplasm in living cells. The technique finds an important field of application in large cells such as oocytes where, during their growth, several organelles and molecules are displaced in specific territories of the cell instrumental for later embryonic development. To measure cell mechanics, cells are usually deformed by many techniques that are slow and often invasive. To overcome these limits, the MPT technique is applied. Probe particles are embedded in the viscoelastic sample and their properties are extracted from the thermal fluctuation spectra measured using digital video-microscopy. The Brownian motion of a probe particle immersed in a network is directly related to the networks mechanical properties. Particles exhibit larger motions when their local environments are less rigid or less viscous. The mean-square-displacement (MSD) of the particles trajectory is used to quantify its amplitude of motions over different time scales.

α-spectrin has a stage-specific asymmetrical localization during Xenopus oogenesis

Xenopus oocyte organization largely depends upon the cytoskeleton distribution, which is dynamically regulated during oogenesis. An actin‐based cytoskeleton is present in the cortex starting from stage 1. At stages 4–6, a complex and polarized cytoskeleton network forms in the cytoplasm. In this paper, we studied the distribution of spectrin, a molecule that has binding sites for several cytoskeletal proteins and is responsible for the determination of regionalized membrane territories. The localization of α‐spectrin mRNA was analyzed during Xenopus oogenesis by in situ hybridization on both whole mount and sections, utilizing a cDNA probe encoding a portion of Xenopus α‐spectrin. Furthermore, an antibody against mammalian α‐spectrin was used to localize the protein. Our results showed a stage‐dependent mRNA localization and suggested that spectrin may participate in the formation of specific domains in oocytes at stages 1 and 2 and 4–6. Mol. Reprod. Dev. 55:229–239, 2000.

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

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.

Following passage through the oviduct, the coelomic envelope of Discoglossus pictus (amphibia) acquires fertilizability upon reorganization, conversion of gp 42 to gp 40, extensive glycosylation, and formation of a specific layer.

This paper describes the morphological and biochemical changes in Discoglossus pictus coelomic oocyte envelope (CE) following passage through the oviduct. As in other anurans, in this species, the transformation of the envelope into vitelline envelope (VE) leads to the acquisition of fertilizability and involves the cleavage of a glycoprotein. In addition, several features, typical of Discoglossus pictus, were observed. A new layer, VE‐D, forms underneath the VE region facing the site of sperm entrance, the dimple. In the VE, arrowhead‐like bundles of fibrils are perpendicularly oriented toward the dimple. Ultrastructural observations and staining with UEA‐I suggested that VE‐D might have a role in supporting sperm penetration into the dimple by orienting VE bundles and exposing sugar residues such as fucose. In ‘in vitro’ tests, VE binding of sperm occurs only if sperm are exposed to A23187, in agreement with previous data (Campanella et al., 1997 : Mol Reprod Dev 47:323–333). Sperm binding occurs all over the VE. Accordingly, extracts of the VE covering the animal or the vegetal hemisphere have the same affinity to lectins (DBA, DSA, GNA, MAA, SBA, SNA, UEA‐I, WGA). The CE contains six main glycoproteins. Peptide mapping indicated that during CE transformation into VE, gp 42 shifts to an apparent Mr of 40 and gp 61 is converted to an apparent Mr of 63 kDa. Lectin blot analyses showed extensive changes in cross‐reactivity of most glycoproteins during the CE→VE transition. The fact that DBA and UEA‐I stain gp 63 rather than gp 61 and that this change is related only to gp 63, suggested that O‐glycosylation and terminal fucose might be acquired by gp 63 in preparation of fertilization. Gp 63 has recently been cloned (Vaccaro et al., submitted) and shown to exhibit high homology to Xenopus gp 69/64, a VE sperm ligand (Tian et al., 1997a : J. Cell Biol. 136: 1099–1108; Tian et al., 1997b : Dev Biol 187:143–153), and to ZP2 of mammals. Mol. Reprod. Dev. 58:318–329, 2001.