Preservation of mouse ovarian tissue follicle morphology and ultra-structure after vitrifying in biotechnological protocols

Abstract

BackgroundThe aim of the present study was to characterize the morphological and ultrastractural of mouse ovarian tissue with different cryoprotectant solution.ObjectiveAim of this study, is to demonstrae an improved convetional vitrification method on mouse ovarian tissue using different concentrations of ethylene glycol (EG) and/or dimetyl sulfoxide (DMSO) and EG.Materials and methodsMouse ovarian tissue dissected and were randomly assigned to three groups: control, conventional vitrification (CV) and toxicity test. Then ovaries were vitrified by 5%, 10% EG or DMSO CV1-CV4, 5%, 10% EG plus DMSO CV5-CV6 and EG plus DMSO in climbing concentrations CV7. The effect of cryoprotectant solutions on ovarian tissue were evaluated by histological examination hematotoxillin & eosin stain, H&E, viability assessment trypan blue stain and ultrastructural analyses transmission electron microscopy, TEM.ResultsOvarian tissue frozen in CV7 solution showed a higher percentage of morphologically normal follicles or viable follicles than other cryoprotectant solutions P < 0.05. Ultrastructural analysis of ovarian tissue showed that less damage was observed in CV7 and it was very similar to the control group.ConclusionVitrification of ovarian tissue with optimal cryoprotectant solutions such as EG plus DMSO is the most effective for preserving the structural efficiency of ovarian follicles.

Supplementation of culture media with vitamin E improves mouse antral follicle maturation and embryo development from vitrified ovarian tissue

“…The second successful method is the addition of a macromolecule, such as sucrose, in equilibration and vitrification solutions. The addition of sucrose confirmed previous reports that this is more efficient for follicular viability and ultrastructure . Sucrose has high solubility and low viscosity, which could facilitate the exit of water from the cell and decrease the formation of ice crystals; thus, it could protect the cells during the freezing–warming process.…”

“…The first successful method for vitrifying mouse ovarian tissue is addition of EG as permeable agents. Our previous study confirmed that EG in combination with DMSO had the highest protective effects and least ultrastructural damage on the ovarian follicle . Thus, in the present study we used mixed cryoprotectants (EG and DMSO) for ovarian tissue vitrification.…”

“…In agreement with previous findings, this study indicated that optimum survival can be attained with 10‐min equilibration in 10% EG + DMSO with 20% sucrose (VS2) and vitrification in 15% EG + DMSO with 20% sucrose (VS3) for ovarian tissue. This allows sufficient time for the cryoprotectant to permeate into the granulosa cells and results in a high proportion of oocytes of normal morphology, fertilization and development to two‐cell embryos and blastocysts after thawing and dilution . However, in our study, no significant difference was observed in the survival, fertilization and developmental rate of frozen–thawed ovarian tissue.…”

“…Thus, in this study, the ultrastructure of vitrified ovaries was analyzed for assessment of cryoprotectant solutions. We concluded in our previous investigation that the morphological integrity and ultrastructure of follicles were preserved after ovarian tissue vitrification with ascending concentration of EG and DMSO . In this regard, step‐by‐step vitrification in ascending concentration of EG and DMSO was used for ovarian tissue cryopreservation.…”

“…Several studies have reported that low molecular weight cryoprotectant agents, such as ethylene glycol (EG) and dimethylsulfoxide (DMSO), could be useful solutions for preservation of ovarian tissue . Vitrification causes changes in the structure of the membrane and follicles' mitochondria . Thus, in this study, the ultrastructure of vitrified ovaries was analyzed for assessment of cryoprotectant solutions.…”