Cytoskeleton structure, pattern of mitochondrial activity and ultrastructure of frozen or vitrified sheep embryos

Abstract

Even though sheep embryo cryopreservation is a commonly used procedure the survival and pregnancy outcomes can vary greatly. This study investigated whether cryopreservation was causing subtle changes in ultrastructure, mitochondrial activity or cytoskeletal integrity. Sheep embryos were either slow cooled in 1.5 M EG (n = 22), or vitrified in 20% EG + 20% DMSO with 0.5 M sucrose in Open Pulled Straws (OPS) (n = 24). One hour after warming the cryopreserved embryos differed from control embryos in that they had no mitochondrial activity combined with cytoskeletal disorganization and large vesicles. Vitrified embryos also showed many points of cytoskeleton disruption. Ultrastructural alterations resulting from actin filaments disorganization were observed in both cryopreserved groups. This includes areas presenting no cytoplasmic organelles, Golgi complex located far from the nucleus and a decrease of specialized intercellular junctions. Additionally, large vesicles were observed in vitrified morulae and early blastocysts. The alterations after cryopreservation were proportional to embryo quality as assessed using the stereomicroscope. Even in the absence of mitochondrial activity, grade I and II cryopreserved embryos contained mitochondria with normal ultrastructure. Embryos classified as grade I or II in the stereomicroscope revealed mild ultrastructural alterations, meaning that this tool is efficient to evaluate embryos after cryopreservation.

Effect of Antioxidants and Apoptosis Inhibitors on Cryopreservation of Murine Germ Cells Enriched for Spermatogonial Stem Cells

“…ATP usage was evaluated because previous work has reported that the mechanism of action of hypotaurine as a cryoprotectant is to increase mitochondrial activity, presumably improving viability and integrity of the cell [31, 32]. In the present study, the effects of various cryoprotective agents on mitochondrial activity mirrored the results of the proliferation analysis.…”

Cryopreservation of Sheep Produced Embryos – Current and Future Perspectives

“…However, in cryopreserved IVP sheep embryos, their function is reduced or absent [2]. Cryopreserved embryos also show cytoskeleton disturbance with actin microfilament disorganization [2,19] that can be caused by cryoprotectants [190]. The ultrastructural evaluation of cryopreserved IVP blastocysts confirmed the presence of more severe cellular damage in these embryos compared to those produced in vivo [18], pointing out the need of research to improve IVP sheep embryo quality and cryotolerance.…”

“…Mitochondria are important for embryo development and metabolism [108,178,190] and thus can be a predictor of the embryo development potential [17,187]. However, in cryopreserved IVP sheep embryos, their function is reduced or absent [2]. Cryopreserved embryos also show cytoskeleton disturbance with actin microfilament disorganization [2,19] that can be caused by cryoprotectants [190].…”

“…Bettencourt et al [187] described the ultrastructure of in vivo-produced sheep embryos, and previously, there were also some insights about the effects of cryopreservation in this source of embryos [17,19,188,189]. Concerning IVP sheep embryos, Dalcin et al [2] revealed its ultrastructure as well as subtle changes in mitochondrial activity or cytoskeletal integrity caused by the cryopreservation process. Later on, Romão et al [18] performed the ultrastructural characterization of fresh and vitrified in vivo and IVP sheep embryos.…”

“…In the last decade, the production of sheep embryos did not get the improvement researchers would like to announce, and its application is still lower compared to other species [1,2]. It is very clear, however, that these techniques will have a central role in animal production in the next few years as a consequence of genetic and reproductive challenges.…”