L. O. Leme

Effects of Different Maturation Systems on Bovine Oocyte Quality, Plasma Membrane Phospholipid Composition and Resistance to Vitrification and Warming

The objective of this study was to evaluate the effects of different maturation systems on oocyte resistance after vitrification and on the phospholipid profile of the oocyte plasma membrane (PM). Four different maturation systems were tested: 1) in vitro maturation using immature oocytes aspirated from slaughterhouse ovaries (CONT; n = 136); 2) in vitro maturation using immature oocytes obtained by ovum pick-up (OPU) from unstimulated heifers (IMA; n = 433); 3) in vitro maturation using immature oocytes obtained by OPU from stimulated heifers (FSH; n = 444); and 4) in vivo maturation using oocytes obtained from heifers stimulated 24 hours prior by an injection of GnRH (MII; n = 658). A sample of matured oocytes from each fresh group was analyzed by matrix associated laser desorption-ionization (MALDI-TOF) to determine their PM composition. Then, half of the matured oocytes from each group were vitrified/warmed (CONT VIT, IMA VIT, FSH VIT and MII VIT), while the other half were used as fresh controls. Afterwards, the eight groups underwent IVF and IVC, and blastocyst development was assessed at D2, D7 and D8. A chi-square test was used to compare embryo development between the groups. Corresponding phospholipid ion intensity was expressed in arbitrary units, and following principal components analyses (PCA) the data were distributed on a 3D graph. Oocytes obtained from superstimulated animals showed a greater rate of developmental (P<0.05) at D7 (MII = 62.4±17.5% and FSH = 58.8±16.1%) compared to those obtained from unstimulated animals (CONT = 37.9±8.5% and IMA = 50.6±14.4%). However, the maturation system did not affect the resistance of oocytes to vitrification because the blastocyst rate at D7 was similar (P>0.05) for all groups (CONT VIT = 2.8±3.5%, IMA VIT = 2.9±4.0%, FSH VIT = 4.3±7.2% and MII VIT = 3.6±7.2%). MALDI-TOF revealed that oocytes from all maturation groups had similar phospholipid contents, except for 760.6 ([PC (34:1) + H]+), which was more highly expressed in MII compared to FSH (P<0.05). The results suggest that although maturation systems improve embryonic development, they do not change the PM composition nor the resistance of bovine oocytes to vitrification.

Molecular markers for oocyte competence in bovine cumulus cells

This study aimed to quantify the expression of candidate genes in cumulus cells (CCs) from cumulus-oocyte complexes (COCs) with high and low potential for in vitro development up to the blastocyst stage. First, the effects of individual culture and biopsy on embryo development were evaluated. Individuals cultured using the well of the well system were compared with individuals cultured in 20 μL droplets (microdroplets) and those cultured in groups (control). Blastocyst rates were lower for the individual culture systems (P < 0.05; well of the well = 17.9%, n = 95; microdrop = 26.3%, n = 95) than for the control group (45.0%, n = 209). Second, the effects of biopsy on embryo production were compared between the control and microdroplet cultures, and no effects (P > 0.05) were observed for either group. Finally, the expression profiles of glypican 4 (GPC4), IGF4-binding protein, follicle-stimulating hormonereceptor, growth hormone receptor, epidermal growth factor receptor, fibroblast growth factor 11, solute carrier family 2 member 1, solute carrier family 2 member 3,sprouty homolog 1, versican, and keratin protein 8 in CCs obtained by biopsy were quantified by real-time polymerase chain reaction. Cumulus cells were categorized on the basis of the fates of the COCs: expanded blastocyst, cleaved and arrested, and uncleaved. The GPC4 gene was overexpressed (P = 0.007) in CCs from oocytes that formed embryos compared with those that produced cleaved and arrested embryos. We concluded that individual culture reduced blastocyst production; however, biopsy did not affect embryo development. The profile of GPC4 expression can be used as a marker to distinguish COCs with potential for embryo development from those with limited developmental potential.

Effect of vitrification using the Cryotop method on the gene expression profile of in vitro–produced bovine embryos

The present study analyzed the changes in gene expression induced by the Cryotop vitrification technique in bovine blastocyst-stage embryos, using Agilent EmbryoGENE microarray slides. Bovine in vitro-produced embryos were vitrified and compared with nonvitrified (control) embryos. After vitrification, embryos were warmed and cultured for an additional 4 hours. Survived embryos were used for microarray analysis and quantitative polymerase chain reaction (qPCR) quantification. Survival rates were higher (P < 0.05) in the control embryos (100%) than in the vitrified embryos (87%). Global gene expression analysis revealed that only 43 out of 21,139 genes exhibited significantly altered expression in the vitrified embryos compared to the control embryos, with a very limited fold change (P < 0.05). A total of 10 genes were assessed by qPCR. Only the FOS-like antigen 1 (FOSL1) gene presented differential expression (P < 0.05) according to both the array and qPCR methods, and it was overexpressed in vitrified embryos. Although, the major consequence of vitrification seems to be the activation of the apoptosis pathway in some cells. Indeed, FOSL1 is part of the activating protein 1 transcription factor complex and is implicated in a variety of cellular processes, including proliferation, differentiation, and apoptosis. Therefore, our results suggest that a limited increase in the rate of apoptosis was the only detectable response of the embryos to vitrification stress.