95 FIRST REPORT ON CLEAVAGE DEVELOPMENT FOLLOWING CRYOPRESERVATION OF ADULT AND PREPUBERTAL RHESUS MONKEY (MACACA MULATTA) OOCYTES

Abstract

Cryopreservation of non-human primate oocytes would allow a more efficient management of biological resources for medical research and endangered species preservation. Despite previous attempts, no cleavage-stage development has been reported following cryopreservation of rhesus monkey (Macaca mulatta) or other non-human primate oocytes. The extreme chilling sensitivity of rhesus monkey oocytes (Songsasen N et al., 2002 Fertil. Steril. 77, 818–825) might be overcomed by vitrification methods. Our aim was to test the Solid Surface Vitrification (SSV, Dinnyes A et al., 2000 Biol. Reprod. 63, 513–518) method on metaphase II (MII)-stage rhesus monkey oocytes and to achieve successful fertilization of warmed oocytes. Oocytes were obtained from hormonally stimulated adult and unstimulated prepubertal females and matured in vitro for 36 to 48 h as described by Zhang P et al. (2001 Biol. Reprod. 64, 1417–1421). The vitrification solution contained 35% ethylene glycol (EG), 5% polyvinyl pyrrolidone, and 0.4 M trehalose in Tyrode-lactate (TL)-HEPES medium with 3 mg mL⁻¹ BSA added. Oocytes at MII-stage were equilibrated in 4% EG in TL-HEPES at 37°C for 10 to 12 min and then exposed to the vitrification solution at 37°C for about 20 s. Oocytes in 2-μL droplets of vitrification solution were dropped onto a metal surface at about −180°C where they were vitrified instantaneously. Warming was performed by moving the vitrified droplets into 0.3 M trehalose at 37°C. Recovered oocytes were exposed to 0.15 M and then 0.075 M trehalose for 2 min each and then rinsed three times in TL-HEPES. Warmed oocytes were fertilized in vitro and then cultured for 96 h in 50-μL drops of mCMRL-1066 containing 20% FCS at 37°C in a humidified atmosphere of 5% CO₂, 5% O₂, and 90% N₂, as described in details by Zhang (see above). A total of 21 MII oocytes were collected from 2 adult and 55 MII oocytes from 14 prepubertal animals. No difference has been found between the rate of adult-origin (15/19; 79%) v. prepubertal-origin (37/52; 71%) oocytes surviving the vitrification process without lysis (P > 0.05, chi-square analyses). Following subsequent IVF, 1/15 (7%) adult-origin and 4/23 (17%) prepubertal-origin oocytes cleaved further, which was lower than that of the corresponding controls (1/2 (50%) and 1/3 (33%), respectively). The furthest development observed following cryopreservation was 16-cell stage, verified by counting of stained nuclei. This is the first report on cleavage-stage development of cryopreserved rhesus monkey oocytes, demonstrating the feasibility of vitrification and the potential for gene banking of non-human primate oocytes, even from prepubertal animals. Further experiments are needed to achieve higher rates of cryosurvival and progeny development. This research was supported by a Chinese-Hungarian Bilateral Technological and Scientific Collaboration Project (No. CHN14/02).