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Stimulation of full-grown amphibian oocytes with progesterone initiates a nontranscriptional signaling pathway that converges in the activation of Cdc2/cyclin B and reentry into meiosis. We observed that cholesterol depletion mediated by methyl-beta-cyclodextrin (MbetaCD) inhibited meiotic maturation, suggesting involvement of membrane rafts. In the present study, we further characterized caveolae-like membranes from Rhinella arenarum oocytes biochemically and functionally. The identification by mass spectrometry of a nonmuscle myosin heavy-chain associated with caveolar membranes showed evidence of direct involvement of the underlying cytoskeletal environment in the structure of oocyte rafts. Biophysical analysis using the fluorescent probe Laurdan revealed that MbetaCD-mediated cholesterol depletion affected membrane lipid order. In line with this finding, cholesterol removal also affected the localization of the raft marker lipid GM1. Results demonstrated that ceramide is an effective inducer of maturation that alters the distribution of the raft markers caveolin-1, SRC, and GM1, while progesterone seems not to affect membrane microdomain integrity. Cholesterol depletion had a greater effect on ceramide-induced maturation, thus suggesting that ceramide is an inducer more vulnerable to changes in the plasma membrane. MbetaCD treatment delayed tyrosine phosphorylation and MAPK activation in progesterone-induced maturation. Functional studies regarding tyrosine phosphorylation raise the possibility that the hormone receptor is located in the nonraft membrane in the absence of ligand and that it translocates to the caveola when it binds to progesterone. The presence of raft markers and the finding of signaling molecules from MAPK cascade functionally associated to oocyte light membranes suggest that this caveolae-rich fraction efficiently recreates, in part, maturation signaling.

The conditions for in vitro oocyte maturation impact on cytoplasmic and nuclear processes in the oocyte. These events are differentially influenced by the nature of the maturation inducer and the presence of intact cumulus in cumulus-oocyte complexes. Epidermal growth factor is the main growth factor promoting oocyte maturation. Also, hyaluronic acid (HA) produced by cumulus cells is known to be responsible for the correct structural and functional organization of the cumulus during oocyte maturation. Therefore, we evaluated the developmental competence of bovine oocytes matured in vitro in a maturation medium supplemented with both EGF and HA, compared to FSH and fetal bovine serum (FBS). In addition, the impact of IVM conditions on the proteomic profile of metaphase II bovine oocytes was analyzed by two-dimensional electrophoresis. Cumulus-oocyte complexes were matured in two media: (1) 10 ng/mL EGF, 15 μg/mL HA, and 100-μM cysteamine and (2) 0.01 UI/mL rh-FSH and 10% FBS. The percentages of first polar body and embryo production and the kinetics of embryo development and oocyte proteomic profiles were analyzed. Oocytes matured in the presence of EGF-HA showed an increase (6%, P 0.05) in the percentage of polar body extrusion. The blastocyst rate was 3% (P 0.05) higher in the FSH-FBS group, but no differences were found in the rate of expanded blastocyst neither in total embryo production between IVM conditions. Cleavage rate of oocytes matured with FSH-FBS was 5% higher (P 0.05) with respect to EGF-HA-matured oocytes when evaluated 30 hours after fertilization. However, at Day 7, those inseminated oocytes that underwent division at a correct timing showed that although there are still early blastocysts in the FSH-FBS condition, EGF-HA embryos have developed completely into blastocysts. Still, the production rate of those embryos that achieved expansion was similar between both maturation conditions. On the other hand, noncleaved presumptive zygotes at Day 7 developed into the different stages with similar rates (∼4%) independently of the medium condition. Modifications of IVM medium composition markedly affected protein profile of bovine oocytes in a differential manner. The proteomic approach revealed the presence of 68 spots in both treatments, 41 exclusively found in the FSH-FBS group and 64 exclusive for the EGF-HA group. Taken together, these results indicate that combined EGF-HA supplementation of in vitro maturation medium could be used to improve oocyte meiotic competence and ensure a better timing to develop into the blastocyst stage.

Linoleic acid (LA) is a polyunsaturated fatty acid present in high concentrations in bovine follicular fluid; when added to maturation culture media, it affects oocyte competence (depending on the type and concentration of LA used). To date, little is known about the effective level of incorporation of LA and there is apparently no information regarding its esterification into various lipid fractions of the oocyte and its effect on neutral lipid storage. Therefore, the objective was to assess the uptake and subcellular lipid distribution of LA by analyzing incorporation of radiolabeled LA into oocyte polar and neutral lipid classes. The effects of various concentrations of LA on the nuclear status and cytoplasmic lipid content of bovine oocytes matured in vitro was also analyzed, with particular emphasis on intermediate concentrations of LA. Neutral lipids stored in lipid droplets were quantified with a fluorescence approach. Linoleic acid at 9 and 43 μM did not affect the nuclear status of oocytes matured in vitro, and 100 μM LA inhibited germinal vesicle breakdown, resulting in a higher percentage of oocytes arrested at the germinal state (43.5 vs. 3.0 in controls; P < 0.05). Bovine oocytes actively incorporated LA from the maturation medium (83.4 pmol LA per 100 oocytes at 22 hours of incubation; P < 0.05) and metabolized it mainly into major lipid classes, e.g., triacylglycerols and phospholipids (61.1% and 29.3%, respectively). Supplementation of the maturation medium with LA increased triacylglycerol accumulation in cytoplasmic lipid droplets at all concentrations assayed (P < 0.05). In conclusion, LA added to a defined maturation medium at concentrations that did not alter the nuclear status of bovine oocytes matured in vitro (9 and 43 μM) improved their quality by increasing the content of neutral lipids stored in lipid droplets. By directing the free fatty acid (LA) to triacylglycerol synthesis pathways and increasing the degree of unsaturation of membrane phospholipids, the oocyte was protected from lipotoxic effects (with an expectation of improved cryotolerance). Fil: Carro, Maria de Las Mercedes. Instituto Nacional de Tecnologia Agropecuaria. Centro Reg.buenos Aires. Estacion Exptal.agrop.balcarce. Laboratorio de Biotecnologia de la Reproduccion; Argentina Fil: Buschiazzo, Jorgelina. Instituto Nacional de Tecnologia Agropecuaria. Centro Reg.buenos Aires. Estacion Exptal.agrop.balcarce. Laboratorio de Biotecnologia de la Reproduccion; Argentina. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina Fil: Rios, Glenda Laura. Instituto Nacional de Tecnologia Agropecuaria. Centro Reg.buenos Aires. Estacion Exptal.agrop.balcarce. Laboratorio de Biotecnologia de la Reproduccion; Argentina Fil: Oresti, Gerardo Martin. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina Fil: Alberio, R. H.. Instituto Nacional de Tecnologia Agropecuaria. Centro Reg.buenos Aires. Estacion Exptal.agrop.balcarce. Laboratorio de Biotecnologia de la Reproduccion; Argentina

Drastic membrane reorganization occurs when mammalian sperm binds to and fuses with the oocyte membrane. Two oocyte protein families are essential for fertilization, tetraspanins and glycosylphosphatidylinositol-anchored proteins. The firsts are associated to tetraspanin-enriched microdomains and the seconds to lipid rafts. Here we report membrane raft involvement in mouse fertilization assessed by cholesterol modulation using methyl-β-cyclodextrin. Cholesterol removal induced: (1) a decrease of the fertilization rate and index; and (2) a delay in the extrusion of the second polar body. Cholesterol repletion recovered the fertilization ability of cholesterol-depleted oocytes, indicating reversibility of these effects. In vivo time-lapse analyses using fluorescent cholesterol permitted to identify the time-point at which the probe is mainly located at the plasma membrane enabling the estimation of the extent of the cholesterol depletion. We confirmed that the mouse oocyte is rich in rafts according to the presence of the raft marker lipid, ganglioside GM1 on the membrane of living oocytes and we identified the coexistence of two types of microdomains, planar rafts and caveolae-like structures, by terms of two differential rafts markers, flotillin-2 and caveolin-1, respectively. Moreover, this is the first report that shows characteristic caveolae-like invaginations in the mouse oocyte identified by electron microscopy. Raft disruption by cholesterol depletion disturbed the subcellular localization of the signal molecule c-Src and the inhibition of Src kinase proteins prevented second polar body extrusion, consistent with a role of Src-related kinases in fertilization via signaling complexes. Our data highlight the functional importance of intact membrane rafts for mouse fertilization and its dependence on cholesterol. Fil: Buschiazzo, Jorgelina. Universite Paris Descartes; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina Fil: Ialy Radio, Come. Universite Paris Descartes; Francia Fil: Auer, Jana. Universite Paris Descartes; Francia Fil: Wolf, Jean Philippe. Universite Paris Descartes; Francia Fil: Serres, Catherine. Universite Paris Descartes; Francia Fil: Lefèvre, Brigitte. Universite Paris Descartes; Francia Fil: Ziyyat, Ahmed. Universite Paris Descartes; Francia

Yolk platelets, the principal components of amphibian oocytes, have been generally considered as material reservoirs. Their biochemical composition and function during oogenesis and early development have not been fully elucidated. The aim of this study was to carry out a lipidic characterization of yolk platelets from full-grown Bufo arenarum oocytes. Ovarian oocytes were manually obtained and the subcellular fraction was isolated by centrifugation at low velocity. Lipids were separated by thin-layer chromatography. For compositional analysis, they were derived by methanolysis, being identified and quantified in a gas-liquid chromatograph. Phospholipid content indicates that phosphatidylcholine and phosphatidylethanolamine are the main phospholipids followed by phosphatidylinositol, sphingomyelin, phosphatidylserine, and phosphatidic acid. Phospholipidic profile is similar to that in whole oocytes except for the absence of dipho- sphatidylglycerol in yolk platelets. Oleic, palmitic, and linoleic acids are the main fatty acids in phosphatidylcholine, and oleic acid is the principal one in phosphatidylethanolamine. In phosphatidic acid, palmitic, estearic, palmitoleic, and oleic acids represent 68 mol% of the total acyl groups. Phosphatidylinositol, enriched in arachidonic acid, is the most unsaturated phospholipid while sphingomyelin shows the lowest unsaturation index. The acyl group distribution in triacylglycerols is similar when yolk platelets and whole oocytes are compared. Polar and neutral lipids of yolk platelets determine the lipidic profile of the whole oocyte. The presence of unusual fatty acids as 14:0, 15:0, 15:1, 17:0, and 17:1 in phospholipids and triacylglycerols may indicate an oxidation mechanism different from b-oxidation in yolk platelets and/or a structural and functional relation with mitochondria. Given that yolk platelets in amphibian oocytes may act in a dynamic fashion in development, their role should be reconsidered. J. Exp. Zool. 297A:189-195, 2003. r 2003

The steroid hormone progesterone induces the resumption of meiosis of full-grown ovarian oocytes arrested at the first meiotic division. Apart from protein-mediated signaling, progesterone-induced maturation involves enzyme activations that modify membrane fluidity and release a cascade of lipid messengers. Once the oocyte is mature, it enters meiosis II and spontaneously arrests in metaphase until fertilization, in which drastic membrane reorganization occurs when amphibian sperm fuses with the oocyte membrane and exocytosis of cortical granules takes place. The predominant lipids in most membranes are phospholipids and cholesterol. Cholesterol stabilizes membranes at low temperatures and it is an important constituent of membrane rafts. In this study, the cholesterol-binding drug methyl- β -cyclodextrin (M β CD) was used for cholesterol modulation in order to assess membrane raft involvement in amphibian fertilization. The generalized polarization (GP) of the fluorescent probe Laurdan was used to study the effect of cholesterol depletion and fertilization on the biophysical properties of membranes from Rhinella arenarum oocytes evaluated in temperatures ranging from 5 °C to 40 °C. For comparative reasons, cholesterol removal of ovarian oocytes and progesterone-induced maturation were also analyzed. As it was expected for native membranes of complex lipid composition, GP values gradually decreased when temperature increased. Light (L) and Heavy (H) membranes, enriched in rafts and non-raft plasma membrane, respectively, showed two different thermotropic profiles of GP. GP values were higher in L fraction with respect to H indicating a higher membrane lipid order in agreement with the enrichment in cholesterol of these low-density membranes. Cholesterol depletion of ovarian oocytes, mediated by M β CD, caused a decrease in the GP value of L membranes which corresponds to a greater penetration of water molecules and indicates a decrease in membrane lipid order. In contrast, GP values of H membranes increased after M β CD treatment. As a result, both plasma membrane fractions become more similar in their biophysical states. On the other hand, progesterone modified the biophysical properties of the plasma membrane increasing the lipid order of both fractions. Interestingly, different to ovarian oocytes, membranes isolated from ovulated oocytes showed an important lipid order resistance to variations in temperature. In these oocytes, M β CD treatment also decreased lipid order of L membranes but at a lesser extent than ovarian oocytes. In addition, fertilization increased lipid order of non-raft membranes. As we have previously shown for progesterone-induce maturation, functional studies revealed that M β CD treatment inhibited fertilization in a dose-dependent manner. Cholesterol repletion performed using M β CD pre-loaded with cholesterol induced a recovery of the fertilization rate. These results indicate that fertilization competence of amphibian oocytes is sensitive to the cholesterol content of both plasma membrane fractions. Taking into account that the plasma membrane is a major site of cryoinjury during cryopreservation of mammalian oocytes, these results open a discussion on sterol modulation to be able to obtain a stable source of oocytes available for in vitro fertilization and other related biotechnologies.

Efficient utilization of bovine oocytes for in vitro embryo production requires successful cryopreservation. However, oocyte development to the blastocyst stage is low after oocyte cryopreservation (Martino et al., 1996). Linoleic acid (LA) is an essential long-chain unsaturated fatty acid (FA), with positive effects reported on cryopreservation. The use of this FA in the culture medium increased the survival rate of frozen-thawed enucleated oocytes and embryos (Hochi et al., 1999, 2000; Tominaga et al., 1999). This effect could be due to an increase in membrane fluidity as a result of the incorporation of an unsaturated FA, preventing their rupture during freezing. According to Pereira and Marques (2008), cold cell damage is mainly due to physical changes experienced by lipids at low temperatures, in particular, intracellular lipids play a major role in the cryosensitivity of oocytes (Nagashima et al., 1995). The aim of this study was to evaluate the effect of LA on intracellular lipid droplets content and its impact on viability of vitrified oocytes. Cumulus oocyte complexes (COC) aspirated from ovaries recovered after slaughter were in vitro matured in a chemically defined maturation medium supplemented with LA at 9, 43 and 100 μM and were denuded and fixed. Oocytes were stained with 1 μg/ml Nile Red in PBS for 10 min. Digital photographs were taken using an epifluorescence microscope and fluorescence intensity was measured with the software Nis Elements Br 3.1. As validated by other authors (Barcelo Fimbres and Seidel 2011), the intensity of fluorescence correlates mainly with the content of triacylglycerols in lipid droplets. Results showed an increase in the mean fluorescence at all concentrations of LA (9, 43 and 100 μM) corresponding to an increase of 17.3, 47.4 and 57.3%, over the control (p