Cortical Vesicles Research Articles

Stereological study of organelle distribution in human mature oocytes

The ultrastructure of human oocytes has been described only qualitatively. To offer a precise organelle spatial distribution and organelle volume during the main maturation stages, we previously conducted stereological studies on prophase-I (GV) and metaphase-I (MI) oocytes, and here we present results on metaphase-II (MII) oocytes. Five donor oocytes from different donors were processed for transmission electron microscopy, and quantification of organelle distribution was performed using point-counting stereology. Statistical tests compared the means of the relative volumes occupied by organelles among oocyte regions. The most abundant organelles were elements of the smooth endoplasmic reticulum (SER), such as SER small vesicles, SER medium vesicles, SER large vesicles and SER isolated tubules, along with mitochondria, followed by SER tubular aggregates, cortical vesicles and lysosomes. Significant differences between oocyte regions were found for lysosomes, cortical vesicles and SER large vesicles. Comparisons of MII oocytes to previous findings in GV and MI oocytes evidenced specific patterns of organelle distribution and relative volumes. This final evaluation thus enables to track organelle spatial reorganization across oocyte stages, which, in addition to gathered knowledge, may be useful to assist in improvements of stimulation protocols, in-vitro maturation media and cryopreservation techniques.

All that glitters is not gold: a stereological study of human donor oocytes

Here we report a quantitative analysis of human metaphase II (MII) oocytes from a 22-year-old oocyte donor, retrieved after ovarian-controlled hyperstimulation. Five surplus donor oocytes were processed for transmission electron microscopy (TEM), and a stereological analysis was used to quantify the distribution of organelles, using the point-counting technique with an adequate stereological grid. Comparisons between means of the relative volumes (Vv) occupied by organelles in the three oocyte regions, cortex (C), subcortex (SC) and inner cytoplasm (IC), followed the Kruskal-Wallis test and Mann-Whitney U-test with Bonferroni correction. Life cell imaging and TEM analysis confirmed donor oocyte nuclear maturity. Results showed that the most abundant organelles were smooth endoplasmic reticulum (SER) elements (26.8%) and mitochondria (5.49%). Significant differences between oocyte regions were found for lysosomes (P = 0.003), cortical vesicles (P = 0.002) and large SER vesicles (P = 0.009). These results were quantitatively compared with previous results using prophase I (GV) and metaphase I (MI) immature oocytes. In donor MII oocytes there was a normal presence of cortical vesicles, SER tubules, SER small, medium and large vesicles, lysosomes and mitochondria. However, donor MII oocytes displayed signs of cytoplasmic immaturity, namely the presence of dictyosomes, present in GV oocytes and rare in MI oocytes, of SER very large vesicles, characteristic of GV oocytes, and the rarity of SER tubular aggregates. Results therefore indicate that the criterion of nuclear maturity used for donor oocyte selection does not always correspond to cytoplasmic maturity, which can partially explain implantation failures with the use of donor oocytes.

The zinc transporter ZIP9 (Slc39a9) regulates zinc dynamics essential to egg activation in zebrafish

The zinc transporter ZIP9 (SLC39A9) was recently characterized as a membrane androgen receptor in various teleost and mammalian cell models. ZIP9 shows the highest expression in ovaries of teleosts, a tissue in which both androgen signaling and zinc dynamics have significant roles. To examine the role of ZIP9 in ovarian physiology, we generated a ZIP9-mutant zebrafish strain using a CRISPR/Cas9 system. zip9-/- females showed significant reductions in fecundity, embryo viability, and growth of their offspring compared to wildtype (WT) fish. Furthermore, a high proportion of zip9-/- eggs failed to undergo normal chorion elevation during activation. In WT eggs, zinc was detected in cortically-localized vesicles which underwent exocytosis upon activation. zip9-/- eggs showed abnormal cortical vesicle development and had a significantly depressed activation-induced zinc release compared to WT eggs. Moreover, pharmacologically sustained elevation of zinc in WT eggs prior to activation resulted in abnormal chorion elevation similar to that observed in zip9-/- eggs. These results indicate that ZIP9 is essential for proper zinc modulation during zebrafish egg activation and presents the first evidence of zinc modulation during egg activation in a non-mammalian species.

SUN-736 Knockout of Membrane Androgen Receptor ZIP9 Results in Reduced Female Fecundity and Abnormal Egg Activation in Zebrafish

Recently, our research group cloned and characterized a putative membrane androgen receptor from teleost ovarian tissue that was found to be homologous with the zinc transporter protein ZIP9 (Slc39a9). To date, ZIP9 is the only zinc transporter that is known to be ligand activated or possess steroid receptor activity. Since the discovery of its androgen receptor activity, ZIP9 has been found to mediate androgen actions in a variety of tissues including teleost ovarian follicle cells, human cancer cell lines, and murine Sertoli cells. However, ZIP9 has not been examined in an in vivo model so the precise physiological functions of this receptor remain unclear. A ZIP9-mutant strain of zebrafish was developed using a CRISPR-Cas9 system in order to examine the role of the protein in teleost reproduction. While ZIP9-mutant males had similar breeding occurrence and fertilization rates to wild-type fish, mutant females exhibited severe reductions in fecundity compared to wild-type fish. ZIP9-mutant females spawn significantly fewer eggs of which a high proportion failed to undergo chorion elevation, a characteristic of normal egg activation. Eggs that showed this failed chorion elevation phenotype had significantly lower fertilization rates and produced larvae that exhibit a high incidence of pericardial/yolk sac edema and reduced growth compared to larvae hatched from wild-type eggs. However, no differences were observed in the proportions of oocytes at later stages of development between ZIP9-mutant and wild-type fish, suggesting the observed phenotypes are not related to abnormal oogenesis. We observed that mature wild-type eggs have numerous cortically located vesicles that are autofluorescent under ultraviolet light and decrease in number when the eggs undergo activation, suggesting they undergo exocytosis during the cortical reaction. While zinc is known to be stored in vesicles that undergo exocytosis in mammalian eggs, the role of zinc in teleost egg activation is currently unknown. In eggs from wild-type fish, we observed an increase in extracellular zinc levels upon egg activation and treatment with a zinc ionophore (zinc pyrithione) significantly reduced the number of eggs that undergo normal chorion elevation when activated. This suggests a role for zinc in zebrafish egg activation similar to that observed in mammals. Of interest, ZIP9-mutant eggs that did not undergo chorion elevation had significantly smaller vesicles than those found in wild-type fish eggs. This abnormal vesicle morphology and failure to undergo chorion elevation suggest a role of ZIP9 in egg activation. Additional insight into the role of zinc in zebrafish egg activation and the mechanism by which ZIP9 disruption leads to abnormal cortical vesicles and egg activation will help determine if ZIP9 plays a role in zinc transport and flux in zebrafish eggs during activation.

Stereological study of organelle distribution in human oocytes at metaphase I.

We have previously presented a stereological analysis of organelle distribution in human prophase I oocytes. In the present study, using a similar stereological approach, we quantified the distribution of organelles in human metaphase I (MI) oocytes also retrieved after ovarian stimulation. Five MI oocytes were processed for transmission electron microscopy and a classical manual stereological technique based on point-counting with an adequate stereological grid was used. Kruskal-Wallis and Mann-Whitney U-tests with Bonferroni correction were used to compare the means of relative volumes (Vv) occupied by organelles. In all oocyte regions, the most abundant organelles were mitochondria and smooth endoplasmic reticulum (SER) elements. No significant differences were observed in Vv of mitochondria, dictyosomes, lysosomes, or SER small and medium vesicles, tubular aggregates and tubules. Significant differences were observed in other organelle distributions: cortical vesicles presented a higher Vv (P = 0.004) in the cortex than in the subcortex (0.96% vs 0.1%) or inner cytoplasm (0.96% vs 0.1%), vesicles with dense granular contents had a higher Vv (P = 0.005) in the cortex than in the subcortex (0.1% vs 0%), and SER large vesicles exhibited a higher Vv (P = 0.011) in the inner cytoplasm than in the subcortex (0.2% vs 0%). Future stereological analysis of metaphase II oocytes and a combined quantitative data of mature and immature oocytes, will enable a better understanding of oocyte organelle distribution during in vivo maturation. Combined with molecular approaches, this may help improve stimulation protocols and in vitro maturation methods.

Altered actin cytoskeleton in ageing eggs of starfish affects fertilization process

Integrity of oocytes is of pivotal interest in the medical and zootechnical practice of in vitro fertilization. With time, oocytes undergo deterioration in quality, and ageing oocytes often exhibit compromised competence in fertilization and the subsequent embryonic development. With ageing oocytes and eggs of starfish (Astropecten aranciacus), we addressed the issue by examining changes of the subcellular structure and their performance at fertilization. Ageing eggs were simulated in two different experimental paradigms: i) oocytes were overmatured by 6 hours stimulation with 1-methyladenine (1-MA); ii) oocytes were removed from the gonad and maintained in seawater for 24 or 48 h before applying the hormonal stimulation (1-MA, 70 min). These eggs were compared with normally matured eggs (stimulated after isolation from the gonad with 1-MA for 70 min) with respect to the sperm-induced intracellular Ca2+ signaling and the structural changes of the egg surface. The cytoskeletal and ultrastructural differences in these eggs were assessed by confocal and transmission electron microscopy, respectively. In the two categories of ageing eggs, we have found remarkable structural modifications of the actin cytoskeleton and the cortical vesicles beneath the plasma membrane. At fertilization, these ageing eggs manifested an altered pattern of intracellular Ca2+ release, aberrant actin dynamics, and increased rate of polyspermy often despite full elevation of the fertilization envelope. Taken together, our results highlight the importance of spatio-temporal regulation of the actin cytoskeleton in the cortex of the eggs, and we postulate that the status of the actin cytoskeleton is one of the major determinants of the oocyte quality that ensures successful monospermic fertilization.

Knockdown of Dinoflagellate Cellulose Synthase CesA1 Resulted in Malformed Intracellular Cellulosic Thecal Plates and Severely Impeded Cyst-to-Swarmer Transition.

Cellulose synthesis (CS) is conducted by membrane-bound cellulose synthase complexes (CSCs), containing cellulose synthases (CesA), that are either arranged in hexagonal structures in higher plants or in linear arrays in most microbial organisms, including dinoflagellates. Dinoflagellates are a major phytoplankton group having linear-type CSCs and internal cellulosic thecal plates (CTPs) in large cortical vesicles. Immunological study suggested CesA1p were cortically localized to the periphery of CTPs. During cyst-to-swarmer transition (TC–S), synchronized peaks of CesA1 transcription, CesA1p expression, CS and CTP formation occurred in respective order, over 12–16 h, strategically allowing the study of CS regulation and CTP biogenesis. CesA1-knockdown resulted in 40% reduction in CesA1p level and time required for swarmer cells reappearance. CTPs were severely malformed with reduced cellulose content. As CTPs are deposited in internal organelle, the present study demonstrated dinoflagellate CesA1 ortholog was adapted for non-surface deposition; this is different to paradigm of other CesAps which require plasmamembrane for cellulose fiber deposition. This pioneer gene-knockdown study demonstrated the requirement of a gene for dinoflagellate cell wall remodeling and proper TC–S, which are prominent in dinoflagellate life-cycles.

Myosins XI Are Involved in Exocytosis of Cellulose Synthase Complexes.

In plants, cellulose is synthesized at the cell surface by plasma membrane (PM)-localized cellulose synthase (CESA) complexes (CSCs). The molecular and cellular mechanisms that underpin delivery of CSCs to the PM, however, are poorly understood. Cortical microtubules have been shown to interact with CESA-containing compartments and mark the site for CSC delivery, but are not required for the delivery itself. Here, we demonstrate that myosin XI and the actin cytoskeleton mediate CSC delivery to the PM by coordinating the exocytosis of CESA-containing compartments. Measurement of cellulose content indicated that cellulose biosynthesis was significantly reduced in a myosin xik xi1 xi2 triple-knockout mutant. By combining genetic and pharmacological disruption of myosin activity with quantitative live-cell imaging, we observed decreased abundance of PM-localized CSCs and reduced delivery rate of CSCs in myosin-deficient cells. These phenotypes correlated with a significant increase in failed vesicle secretion events at the PM as well as an abnormal accumulation of CESA-containing compartments at the cell cortex. Through high-resolution spatiotemporal assays of cortical vesicle behavior, we identified defects in CSC vesicle tethering and fusion at the PM. Furthermore, disruption of myosin activity reduced the delivery of several other secretory markers to the PM and reduced constitutive and receptor-mediated endocytosis. These findings reveal a previously undescribed role for myosin in vesicle secretion and cellulose production at the cytoskeleton-PM-cell wall nexus.

Sphingolipids modulate docking, Ca2+ sensitivity and membrane fusion of native cortical vesicles.

Docking, priming, and membrane fusion of secretory vesicles (i.e. regulated exocytosis) requires lipids and proteins. Sphingolipids, in particular, sphingosine and sphingosine-1-phosphate, have been implicated in the modulation of exocytosis. However, the specific exocytotic steps that sphingolipids modulate and the enzymes that regulate sphingolipid concentrations on native secretory vesicle membranes remain unknown. Here we use tightly coupled functional and molecular analyses of fusion-ready cell surface complexes and cortical vesicles isolated from oocytes to assess the role of sphingolipids in the late, Ca2+-triggered steps of exocytosis. The molecular changes resulting from treatments with sphingolipid modifying compounds coupled with immunoblotting analysis revealed the presence of sphingosine kinase on native vesicles; the presence of a sphingosine-1-phosphate phosphatase is also indicated. Changes in sphingolipid concentrations on vesicles altered their docking/priming, Ca2+-sensitivity, and ability to fuse, indicating that sphingolipid concentrations are tightly regulated and maintained at optimal levels and ratios to ensure efficient exocytosis.

Expression patterns and immunolocalisation of IGF-I and IGF-II in male and female gonads of the Neotropical characid fish Astyanax fasciatus.

The insulin-like growth factor (IGF) system plays important roles in fish reproduction, but the expression pattern and cellular location of IGF-I and IGF-II during gonadal maturation are uncertain. The present study reports a stage-specific assessment of gonadal expression levels and immunolocalisation of IGF-I and IGF-II in Astyanax fasciatus, a characid fish from South America. Adult fish in different maturity stages were caught in the Furnas Reservoir, Grande River, Brazil. Gonad samples were processed for histology, immunohistochemistry, and ELISA for IGF-I and IGF-II. Ovarian levels of IGF-I were low during ripening and ripe stages, higher in totally spent, and then decreased in resting. Levels of IGF-II increased during ovarian maturation, reaching significantly higher values at stage totally spent. In males, IGF-I levels followed gonadal maturation, with higher values in ripening and ripe stages, whereas IGF-II levels showed higher values in stage ripening and partially spent. A positive correlation was found between IGF-I and gonadosomatic index (GSI) for males (r = 0.59), while females showed a negative correlation (r = - 0.43), but IGF-II showed no correlation to GSI. IGF-I was expressed mainly in oogonia nests whereas IGF-II stained the follicular cells in the perinucleolar follicles, cortical vesicles in the previtellogenic follicles, and oogonia nests. In males, IGF-I was evident in spermatogonia and spermatocytes while IGF-II stained Sertoli cells surrounding spermatids cysts and spermatogonia in late stages. Together, these findings support a hypothesis that the balance between IGF-I and IGF-II levels is important in the regulation of gonad maturation in Astyanax fasciatus.

Application of High-Throughput Assays to Examine Phospho-Modulation of the Late Steps of Regulated Exocytosis.

Regulated exocytosis enables a range of physiological functions including neurotransmission, and the late steps (i.e., docking, priming and Ca2+-triggered membrane fusion) are modulated by a highly conserved set of proteins and lipids. Many of the molecular components and biochemical interactions required have been identified; the precise mechanistic steps they modulate and the biochemical interactions that need to occur across steps are still the subject of intense investigation. Particularly, although the involvement of phosphorylation in modulating exocytosis has been intensively investigated over the past three decades, it is unclear which phosphorylation events are a conserved part of the fundamental fusion mechanism and/or serve as part of the physiological fusion machine (e.g., to modulate Ca2+ sensitivity). Here, the homotypic fusion of cortical vesicles was monitored by utilizing new high-throughput, cost-effective assays to assess the influence of 17 small molecule phospho-modulators on docking/priming, Ca2+ sensitivity and membrane fusion. Specific phosphatases and casein kinase 2 are implicated in modulating the Ca2+ sensitivity of fusion, whereas sphingosine kinase is implicated in modulating the ability of vesicles to fuse. These results indicate the presence of multiple kinases and phosphatases on the vesicles and critical phosphorylation sites on vesicle membrane proteins and lipids that directly influence late steps of regulated exocytosis.

New ultrastructural observations of human oocyte smooth endoplasmic reticulum tubular aggregates and cortical reaction: update on the molecular mechanisms involved

IntroductionThe ultrastructure of the human mature metaphase-II oocyte before and after the cortical reaction has been previously described. However, we found a new vesicular aggregate associated with smooth endoplasmic reticulum tubular aggregates (aSERT) and new observations regarding the cortical reaction. ObjectivesTo describe at the ultrastructural level a novel vesicle aggregate associated with aSERT and new observations regarding the cortical reaction. Materials and methodsDonated, surplus mature oocytes were processed for transmission electron microscopy immediately after recover. Calcium detection was performed using the pyroantimonate technique. The cortical reaction was artificially induced by ionophore treatment. ResultsWe observed an accumulation of small vesicles at the periphery of aSERT. At high magnification these were composed by small pale vesicles coated by tiny vesicles, with associated dense materials, giving a rosette-like appearance. Adjacent to these there was another group of small dense vesicles incompletely coated by similar tiny vesicles. Using calcium detection at the ultrastructural level, antimonate deposits were observed in the tubules of aSERT and in the surrounding mitochondria but not in the rosette-like structures. Regarding cortical vesicles, although most previous studies described their contents as homogeneous dense, others referred the presence of another type of cortical vesicles whose contents were moderate dense. Using ionophore oocyte activation, we observed that moderate dense cortical vesicles may correspond to a progressive swelling of the dense cortical vesicles prior to exocytosis. ConclusionsWe describe a novel vesicle aggregate associated to aSERT and new observations on the remodeling of cortical vesicles before exocytosis.

The Role of Phospholipase D in Regulated Exocytosis

There are a diversity of interpretations concerning the possible roles of phospholipase D and its biologically active product phosphatidic acid in the late, Ca(2+)-triggered steps of regulated exocytosis. To quantitatively address functional and molecular aspects of the involvement of phospholipase D-derived phosphatidic acid in regulated exocytosis, we used an array of phospholipase D inhibitors for ex vivo and in vitro treatments of sea urchin eggs and isolated cortices and cortical vesicles, respectively, to study late steps of exocytosis, including docking/priming and fusion. The experiments with fluorescent phosphatidylcholine reveal a low level of phospholipase D activity associated with cortical vesicles but a significantly higher activity on the plasma membrane. The effects of phospholipase D activity and its product phosphatidic acid on the Ca(2+) sensitivity and rate of fusion correlate with modulatory upstream roles in docking and priming rather than to direct effects on fusion per se.

Mammalian gamete fusion depends on the inhibition of ovastacin by fetuin-B.

The zona pellucida, a glycoprotein matrix surrounding the mammalian oocyte, hardens after intrusion of the first spermatozoon, thus protecting the embryo until implantation and preventing multiple fertilizations (polyspermy). Definitive zona hardening is mediated by the metalloprotease ovastacin, which is released from cortical granules of the oocyte upon sperm penetration. However, traces of ovastacin seep from unfertilized eggs to cause zona hardening even in the absence of sperm. These small amounts of protease are inactivated by the plasma protein fetuin-B, thus keeping eggs fertilizable. Once a sperm has penetrated the egg, ovastacin from cortical vesicles overrides fetuin-B and initiates zona hardening.

Allergen Analysis of Sea Urchin Roe Using Sera from Five Patients

Background: Sea urchin roe can cause anaphylactic reactions the first time they are consumed; therefore, careful clinical attention should be paid to their effects. However, no previous study has examined the allergens in sea urchin roe using sera from more than one patient. We attempted to identify sea urchin allergens using sera from 5 patients with sea urchin allergies. Methods: We enrolled 5 patients with relevant medical histories, positive results on a skin prick test and/or a food challenge test, and high levels of sea urchin-specific IgE in an enzyme-linked immunosorbent assay. We performed SDS-PAGE, immunoblotting, immunoblot inhibition, and N-terminal amino acid sequence detection. Results: Ten protein bands ranging from 18 to 170 kDa were detected in more than 2 patients' sera. In immunoblotting, the protein band for the 170-kDa major yolk protein was recognized by 4 of the 5 sera. Furthermore, the reaction between IgE and the protein band for egg cortical vesicle protein (18 kDa) was inhibited by the addition of salmon roe extract. Conclusion: Major yolk protein was confirmed to be one of the main allergens in sea urchin roe. In addition, egg cortical vesicle protein (18 kDa) was demonstrated to be an important protein for cross-reactivity with salmon roe.

Transient fusion and selective secretion of vesicle proteins inPhytophthora nicotianaezoospores

Secretion of pathogen proteins is crucial for the establishment of disease in animals and plants. Typically, early interactions between host and pathogen trigger regulated secretion of pathogenicity factors that function in pathogen adhesion and host penetration. During the onset of plant infection by spores of the Oomycete, Phytophthora nicotianae, proteins are secreted from three types of cortical vesicles. Following induction of spore encystment, two vesicle types undergo full fusion, releasing their entire contents onto the cell surface. However, the third vesicle type, so-called large peripheral vesicles, selectively secretes a small Sushi domain-containing protein, PnCcp, while retaining a large glycoprotein, PnLpv, before moving away from the plasma membrane. Selective secretion of PnCcp is associated with its compartmentalization within the vesicle periphery. Pharmacological inhibition of dynamin function, purportedly in vesicle fission, by dynasore treatment provides evidence that selective secretion of PnCcp requires transient fusion of the large peripheral vesicles. This is the first report of selective protein secretion via transient fusion outside mammalian cells. Selective secretion is likely to be an important aspect of plant infection by this destructive pathogen.

Critical Role of Cortical Vesicles in Dissecting Regulated Exocytosis: Overview of Insights Into Fundamental Molecular Mechanisms

Regulated exocytosis is one of the defining features of eukaryotic cells, underlying many conserved and essential functions. Definitively assigning specific roles to proteins and lipids in this fundamental mechanism is most effectively accomplished using a model system in which distinct stages of exocytosis can be effectively separated. Here we discuss the establishment of sea urchin cortical vesicle fusion as a model to study regulated exocytosis-a system in which the docked, release-ready, and late Ca(2+)-triggered steps of exocytosis are isolated and can be quantitatively assessed using the rigorous coupling of functional and molecular assays. We provide an overview of the insights this has provided into conserved molecular mechanisms and how these have led to and integrate with findings from other regulated exocytotic cells.

Actin Cytoskeleton and Golgi Involvement in Barley stripe mosaic virus Movement and Cell Wall Localization of Triple Gene Block Proteins.

Barley stripe mosaic virus (BSMV) induces massive actin filament thickening at the infection front of infected Nicotiana benthamiana leaves. To determine the mechanisms leading to actin remodeling, fluorescent protein fusions of the BSMV triple gene block (TGB) proteins were coexpressed in cells with the actin marker DsRed: Talin. TGB ectopic expression experiments revealed that TGB3 is a major elicitor of filament thickening, that TGB2 resulted in formation of intermediate DsRed:Talin filaments, and that TGB1 alone had no obvious effects on actin filament structure. Latrunculin B (LatB) treatments retarded BSMV cell-to-cell movement, disrupted actin filament organization, and dramatically decreased the proportion of paired TGB3 foci appearing at the cell wall (CW). BSMV infection of transgenic plants tagged with GFP-KDEL exhibited membrane proliferation and vesicle formation that were especially evident around the nucleus. Similar membrane proliferation occurred in plants expressing TGB2 and/or TGB3, and DsRed: Talin fluorescence in these plants colocalized with the ER vesicles. TGB3 also associated with the Golgi apparatus and overlapped with cortical vesicles appearing at the cell periphery. Brefeldin A treatments disrupted Golgi and also altered vesicles at the CW, but failed to interfere with TGB CW localization. Our results indicate that actin cytoskeleton interactions are important in BSMV cell-to-cell movement and for CW localization of TGB3.

Anionic lipids in Ca2+-triggered fusion

Anionic lipids are native membrane components that have a profound impact on many cellular processes, including regulated exocytosis. Nonetheless, the full nature of their contribution to the fast, Ca2+-triggered fusion pathway remains poorly defined. Here we utilize the tightly coupled quantitative molecular and functional analyses enabled by the cortical vesicle model system to elucidate the roles of specific anionic lipids in the docking, priming and fusion steps of regulated release. Studies with cholesterol sulfate established that effectively localized anionic lipids could contribute to Ca2+-sensing and even bind Ca2+ directly as effectors of necessary membrane rearrangements. The data thus support a role for phosphatidylserine in Ca2+ sensing. In contrast, phosphatidylinositol would appear to serve regulatory functions in the physiological fusion machine, contributing to priming and thus the modulation and tuning of the fusion process. We note the complexities associated with establishing the specific roles of (anionic) lipids in the native fusion mechanism, including their localization and interactions with other critical components that also remain to be more clearly and quantitatively defined.

BIOLOGIA REPRODUTIVA DE FÊMEAS DE LAMBARI-PRATA, Astyanax scabripinnis Jenyns, 1842 (Characidae; Tetragonopterinae; Teleostei) EM CONDIÇÕES DE CATIVEIRO

We studied the reproductive biology of Astyanax scabripinnis by monitoring oocyte development of 94 adult females. Microscopic analysis of the ovaries, gonadosomatic and hepatosomatic indeces assessment, visceral fat and condition factor evaluations were carried out. The structures observed in the ovaries were described from the histological analysis and the hepatosomatic índex, which were used to determine the involvement of the liver in the development of oocytes of this species. Oogonia, oocyte I and oocyte II were present in all stages of gonadal development. Oocytes with cortical vesicles were present in large quantities in the intermediate stage of maturation (F2B) and in a lesser amount in the final stages of maturation (F2C). Mature oocytes were common in the intermediate stage of maturation (F2B), but prevailed in the final stages of maturation (F2C). Oocytes in final maturation and mature viable oocytes, many atresic and post-ovulation folicles were found in spawned animals, besides the ovary disorganization, which are strong characteristics of spawing. Spawned animals were found throughout the year, showing that the species presents a kind of intermittent spawning.KEYWORDS: liver, oocyte; reproduction.