Exogenous progesterone during in vitro fertilization improves developmental competence of partially cumulus-denuded bovine oocytes.

The progesterone (P4) secreted by cumulus cells has gained attention for its role as a possible physiological inducer of sperm acrosome exocytosis. In mammals, it is generally accepted that fertilization rates of oocytes without cumulus are markedly low. This study assessed the integrity of capacitated bovine sperm acrosome when exposed to increasing concentrations of P4, and evaluated whether exogenous P4 during in vitro fertilization (IVF) increases the developmental competence of partially cumulus-denuded oocytes in serum-free conditions. After a 4-h capacitation induction, sperm were incubated with increasing concentrations of P4 (0, 0.1, 10 and 100 µM) and evaluated for viability, caspase activation and acrosome status at three different times (4, 5, and 22 h), including the capacitation induction period. Progesterone induced sperm acrosomal exocytosis without compromising sperm viability or activating sperm caspases. Sperm undergoing acrosome reaction exhibited three differential Concanavalin A patterns, corresponding to early, intermediate and late acrosomal exocytosis. The percentage of these patterns significantly increased over time, regardless of P4 concentration, except for those spermatozoa with late acrosomal exocytosis, which only showed an increase at 22 h of incubation. After incubation for 1 h with 100 µM P4, spermatozoa showing intermediate acrosomal exocytosis significantly increased. At 22 h of incubation, the pattern corresponding to early acrosomal exocytosis evidenced a dose-dependent increase. However, prematurely high levels of acrosome reaction induced by 100 µM P4 led to inefficient IVF outcomes (P < 0.05). Therefore, IVF trials with partially cumulus-denuded oocytes were carried out with lower P4 concentrations (0, 0.1, 1, 5, 10 µM). Cleavage rate significantly increased at 1 µM P4, which translated to increased total embryo production after 7 days of in vitro culture (P < 0.05). Significantly higher percentages of expanded blastocysts were observed at both 1 µM and 10 µM P4 as compared to the other experimental conditions. In conclusion, the different patterns of acrosomal exocytosis identified over time by incubation of live sperm with a fluorescent lectin revealed the existence of sperm subpopulations heterogeneous in their physiological states. Moreover, exogenous P4 at 1 µM during IVF improved the developmental competence of partially cumulus-denuded oocytes in serum-free conditions.

Desmosterol Incorporation Into Ram Sperm Membrane Before Cryopreservation Improves in vitro and in vivo Fertility.

Pregnancy rates in ewes are markedly low after cervical insemination with frozen-thawed sperm. Sensitivity of ram sperm to freeze-thawing is related to the lipid composition of the membrane, particularly to its low sterol content. Recently, we proved that sterol content of ram sperm can be increased by treatment with methyl-ß-cyclodextrin-sterol complexes and we provided mechanistic based evidence on the differential behavior of cholesterol and desmosterol in the ram sperm membrane. In the present study, we evaluated the role of increasing cholesterol and desmosterol content of ram sperm before cryopreservation, on the extent and distribution of sterols, cryocapacitation status, acrosome integrity, DNA damage associated with apoptosis and fertility competence in vitro and in vivo of post-thawed sperm. After freeze-thawing, similar levels of sterol content were evidenced in control sperm cells and in those pre-incubated with either cholesterol or desmosterol. Still, moderately higher levels of sterols were registered in treated sperm compared to the control, indicating no physiological excess of sterols after thawing or sterol losses that exceed the control. Live cell imaging of fluorescent cholesterol evidenced the presence of sperm sub-populations differentially affected by freeze-thawing. Similar unimodal frequency profiles were observed between sterol-enriched groups, while the control exhibited a sub-population of sperm compatible with low sterol content. Tyrosine phosphorylation was significantly lower when ram sperm incorporated cholesterol compared to the control. No difference in this capacitation parameter was found between the latter and desmosterol-enriched sperm. The percentage of sperm with damaged acrosomes post-thawing, assessed by a fluorescent lectin, was reduced in sperm that incorporated sterols before freezing, irrespective of the sterol class. These results suggest that sterols exert a stabilizing effect on the acrosome. No differences were found in levels of apoptotic DNA fragmentation among experimental groups. As to fertility trials, desmosterol-enriched sperm gave rise to higher rates of in vitro activated oocytes by heterologous fertilization and to significantly lower pregnancy loss in vivo. Our research provides new insights on sterol incorporation into ram sperm prior to cryopreservation, in particular on the additional benefit of incorporating desmosterol as a strategy to improve fertility outcome.

Cholesterol and desmosterol incorporation into ram sperm membrane before cryopreservation: Effects on membrane biophysical properties and sperm quality.

Ram sperm are particularly sensitive to freeze-thawing mainly due to their lipid composition, limiting their use in artificial insemination programs. We evaluated the extent of cholesterol and desmosterol incorporation into ram sperm through incubation with increasing concentrations of methyl-ß-cyclodextrin (MßCD)-sterol complexes, and its effect on membrane biophysical properties, membrane lateral organization and cryopreservation outcome. Sterols were effectively incorporated into the sperm membrane at 10 and 25 mM MßCD-sterols, similarly increasing membrane lipid order at physiological temperature and during temperature decrease. Differential ordering effect of sterols in ternary-mixture model membranes revealed a reduced tendency of desmosterol of segregating into ordered domains. Live cell imaging of fluorescent cholesterol showed sterol incorporation and evidenced the presence of sperm sub-populations compatible with different sterol contents and a high concentration of sterol rich-ordered domains mainly at the acrosome plasma membrane. Lateral organization of the plasma membrane, assessed by identification of GM1-related rafts, was preserved after sterol incorporation except when high levels of sterols (25 mM MßCD-desmosterol) were incorporated. Ram sperm incubation with 10 mM MßCD-sterols prior to cryopreservation in a cholesterol-free extender improved sperm quality parameters after cooling and freezing. While treatment with 10 mM MßCD-cholesterol increased sperm motility, membrane integrity and tolerance to osmotic stress after thawing, incorporation of desmosterol increased the ability of ram sperm to overcome osmotic stress. Our research provides evidence on the effective incorporation and biophysical behavior of cholesterol and desmosterol in ram sperm membranes and on their consequences in improving functional parameters of sperm after temperature decrease and freezing.

Free cholesterol and cholesterol esters in bovine oocytes: Implications in survival and membrane raft organization after cryopreservation.

Part of the damage caused by cryopreservation of mammalian oocytes occurs at the plasma membrane. The addition of cholesterol to cell membranes as a strategy to make it more tolerant to cryopreservation has been little addressed in oocytes. In order to increase the survival of bovine oocytes after cryopreservation, we proposed not only to increase cholesterol level of oocyte membranes before vitrification but also to remove the added cholesterol after warming, thus recovering its original level. Results from our study showed that modulation of membrane cholesterol by methyl-ß-cyclodextrin (MßCD) did not affect the apoptotic status of oocytes and improved viability after vitrification yielding levels of apoptosis closer to those of fresh oocytes. Fluorometric measurements based on an enzyme-coupled reaction that detects both free cholesterol (membrane) and cholesteryl esters (stored in lipid droplets), revealed that oocytes and cumulus cells present different levels of cholesterol depending on the seasonal period. Variations at membrane cholesterol level of oocytes were enough to account for the differences found in total cholesterol. Differences found in total cholesterol of cumulus cells were explained by the differences found in both the content of membrane cholesterol and of cholesterol esters. Cholesterol was incorporated into the oocyte plasma membrane as evidenced by comparative labeling of a fluorescent cholesterol. Oocytes and cumulus cells increased membrane cholesterol after incubation with MßCD/cholesterol and recovered their original level after cholesterol removal, regardless of the season. Finally, we evaluated the effect of vitrification on the putative raft molecule GM1. Cholesterol modulation also preserved membrane organization by maintaining ganglioside level at the plasma membrane. Results suggest a distinctive cholesterol metabolic status of cumulus-oocyte complexes (COCs) among seasons and a dynamic organizational structure of cholesterol homeostasis within the COC. Modulation of membrane cholesterol by MßCD improved survival of bovine oocytes and preserved integrity of GM1-related rafts after vitrification.

Cholesterol depletion disorganizes oocyte membrane rafts altering mouse fertilization.

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-ß-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.

Nongenomic steroid- and ceramide-induced maturation in amphibian oocytes involves functional caveolae-like microdomains associated with a cytoskeletal environment.

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.

Inhibition of Bufo arenarum oocyte maturation induced by cholesterol depletion by methyl-beta-cyclodextrin. Role of low-density caveolae-like membranes.

The invaginated structure of caveolae seems to provide an optimal environment for hormone binding leading to oocyte meiotic maturation. We conducted a quantitative analysis of lipids and proteins of detergent-free low-density membranes isolated from Bufo arenarum oocytes and we modulated cellular cholesterol to further understand how these domains perform their regulatory functions in the amphibian system. Light membranes derive from the plasma membrane as suggested by the enrichment in the activity of 5'nucleotidase. Lipid analysis by chromatography techniques revealed that this fraction is enriched in phosphatidylserine and cholesterol and that it evidences an important level of sphingomyelin. The finding of a single 21 kDa caveolin in light membranes indicates the presence of caveolae-like structures in B. arenarum oocytes. In support of this finding, c-Src is significantly associated to this fraction. Cholesterol content of oocytes treated with methyl-beta-cyclodextrin (MbetaCD) decreased when compared to control oocytes. Drug treatment inhibited meiotic maturation in a dose-dependent manner and affected the localization of caveolin and c-Src among membrane fractions. Repletion of cholesterol showed a recovery of the ability of MbetaCD-treated oocytes to mature, particularly at the 25 mM concentration in which reversibility was close to the control level. Results highlight the importance of caveolae-like microdomains for maturation signaling in Bufo oocytes.

Effect of meiotic maturation on yolk platelet lipids from Bufo arenarum oocytes.

Progesterone induces the resumption of meiosis in Bufo arenarum full-grown arrested oocytes through a nongenomic mechanism called meiotic maturation. Growing evidence indicates that lipids are involved in the maturation process. They are mainly located in yolk platelets, the principal organelles of amphibian oocytes. The aim of the present study was to analyze the effect of progesterone-induced maturation on lipids from B. arenarum yolk platelets. Ovarian oocytes, manually obtained, were incubated with progesterone to induce maturation. Yolk platelets were isolated by centrifugation at low velocity. Lipids were separated by thin-layer chromatography. For compositional analysis, they were derivatized by methanolysis, and were identified and quantified in a gas-liquid chromatograph. Phospholipid content decreased in progesterone-treated oocytes, mainly as a result of a decrease at the level of phosphatidylcholine (PC). The turnover of this lipid is considered crucial for the completion of meiosis. Sphingomyelin also underwent a decrease that could be related to the important role of ceramide as an inducer of germinal vesicle breakdown. Maturation effect on fatty acid composition registered significant changes in PC whose saturated fatty acids increased. A net increase in arachidonic acid was observed in phosphatidylserine after progesterone treatment. The contents of total triacylglycerols and diacylglycerols were not significantly modified by hormone effect while free fatty acids underwent a significant increase as a result of polyunsaturated fatty acids increase. Altogether, our results demonstrate that yolk platelet lipids are involved in the resumption of the meiotic cell cycle, thus suggesting that these organelles participate in a dynamic role during amphibian development.

Detailed lipid analysis of yolk platelets of amphibian (Bufo arenarum) oocytes.

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 diphosphatidylglycerol 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 beta-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.

Lipids during Bufo arenarum oogenesis.

The content and composition of phospholipids and triacylglycerols (TAGs) in Bufo arenarum oocytes in stages III and IV of their oogenesis were studied. The total amount of phospholipids in stage IV oocytes is 0.5-fold higher than in stage III oocytes. In both cases, the main phospholipids are phosphatidylcholine (PC) and phosphatidylethanolamine (PE). A striking observation concerns the high level of diphosphatidylglycerol (DPG) in stage III oocytes, which could be indicative of a relatively larger mitochondrial population with respect to other oogenetic stages. A net increase in sphingomyelin content was found during oogenesis. This fact could be related to the role of this phospholipid in the signal transductional pathways. In PC, palmitic (16:0), linoleic (18:2) and oleic (18:1) are the major fatty acids for both types of oocytes, while in PE the main acyl groups are 18:1, 16:0, arachidonic acid (20:4n6) and 18:2. PE is more unsaturated than PC and both phospholipids are more unsaturated in stage III oocytes than in stage IV oocytes. The amount of triacylglycerols is 0.3-fold higher in stage IV oocytes than in stage III oocytes. In both stages, the main fatty acids are 18:2, 18:1 and 16:0. During oogenesis, a significant increase in 18:1 and 18:3n3, and a decrease in 18:2 of TAG were found. The unsaturation index of TAGs from stage IV oocytes is higher than that from stage III oocytes. The TAG increase during oogenesis is consistent with the putative use of these lipids as a source of energy in embryo development.