Optimizing vitrification of in vitro matured bovine oocytes /
- Autores: Núria Arcarons Deseures
- Directores de la Tesis: María Teresa Mogas Amorós (dir. tes.)
- Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2017
- Idioma: español
- Tribunal Calificador de la Tesis: María Teresa Paramio Nieto (presid.), Nuno Luis Costa Borges (secret.), Urban Bessenfelder (voc.)
- Programa de doctorado: Programa Oficial de Doctorado en Medicina y Sanidad Animales
- Materias:
- Enlaces
- Tesis en acceso abierto en: DDD
- Resumen
The numerous benefits of bovine oocyte cryopreservation has dramatically increased the demand of bovine oocytes. In this context, successful preservation of in vitro matured bovine oocytes appears to be critical for guarantee the oocyte supply. In spite of that, an efficient and efficacious vitrification protocol for in vitro matued oocytes should be achieved soon. The purpose of this thesis is, therefore, to investigate the use of high concentrations of NaCl or sucrose prior to vitrification/warming, the oolema enrichment with cholesterol prior to vitrification and the addition of an ice bocking biopolymer synthetized by a bacteria from Antarctica during vitrification and warming, as strategies to improve bovine oocyte vitrification protocols. Exposure of oocytes to increased concentrations of sodium chloride, sucrose or trehalose prior to manipulation has been reported to improve both cryotolerance to vitrification and developmental competence in porcine specie. In the chapter IV of this thesis we observed that treatment with 375 mOsmol NaCl or sucrose solution for 1 h before vitrification had no detrimental effects on the meiotic spindle status of IVM bovine oocytes. In particular, sucrose pretreatment prior to vitrification was unable to improve embryo development as observed in other species.
Membrane cholesterol enrichment could increase the fluidity and permeability of the membrane and increase the cryotolerance of oocytes to cryopreservation. In chapter V of this thesis we used bodipy-cholesterol to image the cholesterol transport in live in vitro matured bovine oocytes incubated with cholesterol-loaded methyl-β-cyclodextrin by confocal microscopy. This method allowed us to determine the incubation time required for optimal cholesterol incorporation into membrane avoiding the non-desired penetration into cytoplasm, using different cholesterol-loaded methyl-β-cyclodextrin concentrations in different supplemented media. However, cryotolerance in terms of survival and developmental competence was not improved, regardless of the application of the determined cholesterol-loaded methyl-β-cyclodextrin treatment or the holding medium used. However, the cholesterol addition before vitrification, altered the expression of genes related to lipid metabolism (CYP51), apoptosis (BAX) and DNA methylation (DNMT3A) in bovine morulae, mainly when oocytes were vitrified at germinal vesicle stage.
Pseudomonas sp. ID1, a bacterium isolated from marine sediment from Antarctica, produces an exopolysaccharide as a cold adaptation mechanism (M1 EPS). This exopolysaccharide conferred cryoprotection for other bacteria cells, suggesting it can thus be applied as an agent for cell cryopreservation. In the Chapter VI the M1 EPS was added to vitrification and warming solutions as an ice blocking agent to limit the in vitro matured bovine oocyte damage and to increase further developmental competence. The effects of different concentrations of M1 EPS supplementation were examined on meiotic spindle organization, developmental competence in terms of blastocyst rates, and gene expression in day 8 bastocyts. M1 EPS supplementation during vitrification and warming of in vitro matured prepubertal heifer oocytes protected the meiotic spindle against chromosome and microtubule decondensation caused by vitrification. Although no improvement on blastocyst rates were observed after EPS supplementation, different changes in gene expression of some gens related with epigenetics (DNMT3A and KAT2A) and blastocyst quality (BAX, BCL2) were recorded between blastocysts derived from oocytes vitrified with different concentrations of M1 EPS.
In summary, on the basis of the results of the present Thesis, we can conclude that in vitro matured oocyte cryotolerance does not only depend on the damage on the meiotic spindle organization, but also on the gene expression changes that determine further embryonic development.
