Cryoprotectant role of exopolysaccharide of Pseudomonas sp. ID1 in the vitrification of IVM cow oocytes.

Biological molecules isolated from organisms that live under subzero conditions could be used to protect oocytes from cryoinjuries suffered during cryopreservation. This study examined the cryoprotectant role of exopolysaccharides of Pseudomonas sp. ID1 (EPS ID1) in the vitrification of prepubertal and adult cow oocytes. IVM oocytes were vitrified and warmed in media supplemented with 0, 1, 10, 100 or 1000µgmL-1 EPS ID1. After warming, oocytes were fertilised and embryo development, spindle morphology and the expression of several genes in Day 8 blastocysts were assessed. Vitrification led to significantly lower proportion of prepubertal oocytes exhibiting a normal spindle configuration. In fresh control oocytes and most groups of vitrified adult oocytes, similar percentages of oocytes with a normal spindle configuration were observed. Percentages of Day 8 blastocysts were similar for prepubertal oocytes vitrified in the absence or presence of 1 or 10µgmL-1 EPS ID1 and for adult oocytes vitrified in the presence of 10µgmL-1 EPS ID1 compared with non-vitrified oocytes. EPS ID1 supplementation had no effect on solute carrier family 2 member 3 (SLC2A3), ubiquitin-conjugating enzyme E2A (UBE2A) and histone deacetylase 1 (HDAC1) expression in Day 8 blastocysts form adult oocytes. However, supplementation with 10 and 100µgmL-1 EPS ID1 led to increased expression of genes involved in epigenetic modifications (DNA methyltransferase 3 alpha (DNMT3A) and K (lysine) acetyltransferase 2A (KAT2A)) and apoptosis (BCL2 associated X apoptosis regulator (BAX) and BCL2-like 1 (BCL2L1)). The lowest BAX:BCL2L1 ratio was found in the 10µgmL-1 EPS ID1-supplemented group. The results suggest that 10µgmL-1 EPS ID1 added to vitrification and warming media may help protect bovine oocytes against cryodamage.

Cholesterol added prior to vitrification on the cryotolerance of immature and in vitro matured bovine oocytes.

This study examines whether incorporating cholesterol-loaded methyl-ß-cyclodextrin (CLC) in the bovine oocyte plasma membrane improves oocyte tolerance to vitrification. In vitro matured oocytes were incubated with 2 mg/ml BODIPY-labeled CLC for different time intervals in FCS or PVA supplemented medium or exposed to different CLC concentrations to examine the subcellular localization of cholesterol by confocal microscopy live-cell imaging. Subsequently, the effects of optimized CLC concentrations and incubation times prior to vitrification on early embryo development were assessed. Then, we evaluated the effects of pretreatment with 2 mg/ml CLC for 30 min before the vitrification of immature (GV) and in vitro matured (MII) oocytes on developmental competence and gene expression. Our results indicate a high plasma membrane labeling intensity after 30 min of incubation with 2 mg/ml CLC for 30 min, regardless of the holding medium used. When oocytes were incubated with 1 mg/ml, 2 mg/ml and 3 mg/ml of CLC, intense labeling was observed at the plasma membrane after 40, 30 and 20 min, respectively. CLC pre-treatment before the vitrification of bovine oocytes did not affect subsequent cleavage and embryo development rates irrespective of CLC concentrations, incubation times or meiotic stage. However, pretreatment seems to improve the quality of embryos derived from vitrified oocytes, mainly when oocytes were vitrified at the GV stage.

Assessment of the effect of adding L-carnitine and/or resveratrol to maturation medium before vitrification on in vitro-matured calf oocytes.

Cryopreservation may lead bovine oocytes to undergo morphological changes and functional damage due to the high-lipid content in the cytoplasm and the formation of reactive oxygen species. Against this background, the present study aimed to improve the cryotolerance and developmental competence of prepubertal calf oocytes by adding L-carnitine (LC) and/or resveratrol (R) to the IVM medium, as the former is involved in lipid metabolism and both are able to scavenge reactive oxygen species. With this purpose, different quality and functional oocyte parameters, such as spindle and chromosome configuration, DNA integrity, caspase activity, and the profile of genes involved in lipid metabolism and oxidative stress were evaluated in IVM bovine oocytes before or after vitrification/warming. Oocytes were matured in the absence (control) or presence of LC (3.03 mM) and/or R (1 µM) and then vitrified/warmed before IVF and embryo culture. All treatment groups (control, LC, R, and LC + R) of nonvitrified IVM oocytes showed similar rates (P > 0.05) of a normal spindle and chromosome configuration to oocytes vitrified/warmed after maturation in the presence of LC + R. When oocytes in all treatment groups were compared before and after vitrification, no significant differences were detected in DNA fragmentation as measured using the TUNEL method. However, the proportion of early apoptotic oocytes increased after vitrification/warming, except when previously matured with R. Vitrified/warmed oocytes matured in the presence of LC did not differ with nonvitrified oocytes in terms of the expression of ACACA, SLC2A1, PLIN2, HSPA1A, GPX1, and SOD1 genes. Similarly, expression of ACACA, SLC2A1, PLIN2, HSPA1A, and SOD1 genes in vitrified/warmed oocytes was similar to that of their fresh counterparts when matured in the presence of R. Finally, while the addition of LC and/or R to IVM medium had no effect on both cleavage and blastocyst rates either in fresh or vitrified oocytes. To conclude, the results of the present study report that the addition of LC and/or R to the IVM medium used for prepubertal bovine oocytes did not increase the embryo development potential of both fresh and vitrified oocytes. However, LC + R supplementation before vitrification decreased spindle damage, R addition-modulated apoptosis, and LC or R addition before vitrification positively affected the gene expression of vitrified/warmed oocytes.

Spindle configuration and developmental competence of in vitro-matured bovine oocytes exposed to NaCl or sucrose prior to Cryotop vitrification.

In the present study we examined whether exposure to high concentrations of NaCl or sucrose before vitrification improves the cryotolerance of in vitro-matured bovine oocytes. In Experiment 1, oocytes were exposed to different concentrations of NaCl (375-1517 mOsm) or sucrose (375-812 mOsm) for 1h. On the basis of the results of this experiment, in Experiment 2 oocytes were exposed to 0.25% NaCl (375 mOsmol) or 2.77% sucrose (375 mOsmol) solution, vitrified and warmed. Microtubule and chromosome configurations were examined by immunocytochemistry. In Experiment 3, in vitro embryo development was assessed after vitrification of oocytes with or without 2.77% sucrose (375 mOsmol) pretreatment. There was a similar percentage of oocytes showing normal spindle configurations in the sucrose-pretreated and control groups. Higher rates of abnormal spindles were found in groups treated with NaCl or sucrose solutions with >375 mOsmol. After vitrification and warming, a significantly higher percentage of oocytes with normal chromosome configurations was recorded for oocytes exposed to 375 mOsmol sucrose solution before vitrification compared with the control vitrified oocytes. However, these percentages were significantly lower than those recorded in untreated controls. Cleavage and blastocyst rates were higher in non-vitrified than vitrified oocytes. In conclusion, pretreatment with 375 mOsmol NaCl or sucrose solution had no adverse effects on the spindle status of vitrified-warmed cow oocytes. However, sucrose pretreatment offered no benefits for embryo development.