Vitrificación de ovocitos porcinos y su efecto a nivel estructural en embriones y en el ADN de las células del cúmulo

Abstract

Vitrification is mainly used to cryopreserve embryos and female gametes. This technique allows maintaining cell viability, functionality, and developmental potential at low temperatures into liquid nitrogen at -196 ºC. For this, the addition of cryoprotectant agents, which are substances that provide cell protection during cooling and warming, is required. However, they have been reported to be toxic, reducing oocyte viability, maturation, fertilization, and embryo development, possibly by altering cell cytoskeleton structure and chromatin. Previous studies have evaluated the effects of vitrification in the germinal vesicle, metaphase II oocytes, zygotes, and blastocysts, but the knowledge of its impact on their further embryo development is limited.Other studies have evaluated the role of actin microfilaments and chromatin, based on the fertilization and embryo development rates obtained, but not the direct evaluation of these structures in embryos produced from vitrified immature oocytes. Therefore, this study was designed to evaluate how the vitrification of porcine immature oocytes affects early embryo development by the evaluation of actin microfilament distribution and chromatin integrity.Results demonstrate that the damage generated by the vitrification of immature oocytes affects viability, maturation, and the distribution of actin microfilaments and chromatin integrity, observed in early embryos, but did not affect in vitro fertilization. Therefore, it is suggested that vitrification could affect oocyte repair mechanisms in those structures, being one of the mechanisms that explain the low embryo development rates after vitrification.On the other hand, the evaluation of the DNA damage generated in cumulus cells after mature cumulus-oocyte complexes vitrification can be considered as an indicator of oocyte quality since these cells play important roles in oocyte developmental competence. Therefore, the aim of this study was to determine if matured cumulus-oocyte complexes exposure to cryoprotectants (CPAs) or vitrification affects oocytes and cumulus cells viability, but also if DNA damage is x generated in cumulus cells, affecting fertilization and embryo development. The DNA damage in cumulus cells was measured using the alkaline comet assay and expressed as Comet Tail Length and Olive Tail Moment (OTM). Results demonstrate that oocyte exposure to CPAs or vitrification reduced oocyte (75.5 ± 3.69 %, toxicity; 66.7 ± 4.57 %, vitrification) and cumulus cells viability (32.7 ± 5.85 %, toxicity; 7.7 ± 2.21 %, vitrification) compared to control (95.5 ± 4.04 %, oocytes; 89 ± 4.24 %, cumulus cells). Also, significantly higher DNA damage expressed as OTM was generated in the cumulus cells after exposure to CPAs and vitrification (39 ± 17.41, 33.6 ± 16.69, respectively) compared to control (7.4 ± 4.22). In addition, fertilization and embryo development rates also decreased after exposure to CPAs (35.3 ± 16.65 %, 22.6 ± 3.05 %, respectively) and vitrification (32.3 ± 9.29 %, 20 ± 1 %, respectively). This stu...