94 VITRIFICATION OF WHOLE OVARIES IN YOUNG RATS

Cryopreservation of reproductive organs is an important technology for preservation of genetic resources of experimental, domestic, and wild animals. In addition, cryopreservation of the ovary could be applied to restore the fertility of young women diagnosed with cancer because it could not only provide future fertility, but could also decrease the emotional consequences of cancer therapy for women afflicted with such devastating diseases. Cryopreservation of whole ovary particularly would enable such females to be pregnant by natural mating after transplantation. The aim of the present study was to evaluate the possible of vitrification for young rat ovaries as a possible model for human ovaries using a Cryotop method. Whole ovaries were collected from 10-day-old female rats (Brown-Norway × Wistar) and then washed several times in PB1 medium containing 20% (v/v) fetal calf serum (FCS). The ovaries were submerged in equilibration solution [10% (v/v) dimethyl sulfoxide (DMSO), 10% (v/v) ethylene glycol (EG), and 20% (v/v) FCS in PB1] for 1, 3, 5 or 10 min and then vitrified in vitrification solution (20% DMSO, 20% EG, and 20% FCS in PB1) for 5 min, being plunged into liquid nitrogen on Cryotops. Vitrified ovaries were later warmed in 37°C in PB1 containing 1 M sucrose and 20% FCS for 5 min and then PB1 containing 0.5 M sucrose and 20% FCS for 5 min. Eight ovaries were embedded in paraffin and cut into 4 μm slices. The slices were stained by hematoxylin-eosin (HE) and integrity of nuclei and cytoplasm in the follicles was histologically evaluated. In each group, 3 ovaries were used for the staining of live/dead (Invitrogen LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells, #L3224). Follicles were separated from the ovary with enzyme and then classified into 2 categories by staining. Follicles possessing more than 50% of green-stained and red-stained granulosa cells were divided as live and dead, respectively. Data were analyzed by Tukey’s test. Results of the HE staining showed that integrity of nuclei and cytoplasm in ovaries equilibrated for 10 min (11%) was lower than those for 1 min (25%), 3min (42%), and 5 min (30%). As for evaluation by live/dead staining, most of follicles in ovaries equilibrated for 10 min (25%) were stained with red (dead). In vitrified ovaries equilibrated for 1 min, 3 min, and 5 min (18%, 16%, and 18%, respectively), there were no significant differences in red-stained follicles. These results suggest that optimal equilibration time is 3 to 5 min for vitrification of whole ovaries of 10-day-old rats. Using this equilibration time, we are now trying to evaluate the capability of vitrified/warmed ovaries after transplantation to recipient rats. The work was supported in part by Grant-in-Aid for Scientific Research from JSPS (KAKENHI) (21789253) to J. I. This work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan through a Grant-in-Aid for Matching Fund Subsidy for Private Universities to J.I. and N.K.