P-99: The Effects of Zinc in Vitrification Medium on In Vitro Growth of Follicles Derived from Vitrified-Warmed Mouse Ovary

Background: The development of a reliable method for the cryopreservation of mammalian ovary would be an important advancement in the field of reproductive biology for the preservation of genetic resources. The cryopreserved follicles have the potential to develop in-vitro; however, the developmental rate is lower than that of fresh follicles. Researchers have used that of different cryoprotectants and various techniques to improve the cryopreservation of ovaries despite significant recent progress, the efficiency of ovary cryopreservation is still low.There are many reports on adding some materials such as calcium, ascorbate and antioxidants to freezing medium. The necessity of inorganic elements in biological systems is well established. Zinc is an integral component of hundreds of enzymes, transcription factors, and other molecules involved in a variety of biological functions, and the homeostasis of this trace element is tightly regulated.Zinc, in particular, has gained attention as key agent in cellular signaling processes. The present study was designed to determine whether different zinc concentrations in the ovary vitrification solutions could improve the developmental growth and competence of follicles derived from vitrified ovaries. Materials and Methods: In this experimental study, the ovaries of 2-4 week-old NMRI mice randomly assigned to following groups :V0(vitrified-warmed ovaries without any zinc in vitrification solution),V1,V2,V3 (vitrified warmed ovaries with 100,150 and 200 μg/ dl zinc concentration in vitrification solution,N-v (none vitrified ovaries). Ovaries in the vitrified groups were vitrified sequentially by immersion into two vitrification solutions VS1: 7.5% ethylene glycol (EG) + 7.5% DMSO in holding medium (α-MEM + 20% FBS) for 7 minutes and VS2: 15% EG + 15% DMSO for 3 minutes in holding medium and vitrified by straw and were keeped in LN2 tank for a week. After one week, the ovaries were thawed at temprature room in 1.0,0.5 and 0.25 M sucrose. Vitrified ovaries as well as non-vitrified ovaries were serially sectioned and examined histologically. Pre-antral follicles (specified with a diameter around 100-130 mm) were mechanically isolated from vitrified-warmed and fresh ovaries and cultured for 9 days. Results: The results show that the presence of zinc in vitrification solution is effective and can reduce the traumatic effects of vitrification. Follicle viability,growth and survival rate was better preserved in the 200 μg/dl zinc concentration group in comparsion to other vitrified-warmed groups. Nevertheless, it was less than that of none vitrified group(Anova, p value<0.05). Vitrification by using EG and DMSO is an efficient procedure for cryopreservation of ovaries. Conclusion: The results of this study demonstrate that zinc supplementation of vitrification medium in a time and dose manner improved the follicle growth in follicles derived from vitrified-warmed ovaries.