79 DEVELOPMENT TO TERM OF RAT ZYGOTES DERIVED FROM CRYOPRESERVED MATURE OOCYTES AND SPERM THROUGH INTRACYTOPLASMIC SPERM INJECTION

Abstract

The rat, as well as the mouse, is one of the most valuable experimental animals for biomedical and physiological research. There are numerous valuable mutant rats including transgenetic strains. Cryopreservation of rat oocytes and sperm as haploid germ cells is a key technology for banking the genetic resources efficiently. The aim of the present study was to examine survival of vitrified/warmed oocytes and developmental competence of resultant zygotes in the rat. Rats used in the present study were all Wistar rats. Epididymal spermatozoa were frozen as described previously (Seita et al. 2005 Reprod. Fertil. Dev. 18, 256). After thawing, spermatozoa were sonicated to obtain sperm heads for intracytoplasmic sperm injection (ICSI). Oocytes were collected from immature females superovulated with eCG and hCG. Oocytes were equilibrated in 7.5% (v/v) ethylene glycol (EG) + 7.5% (v/v) dimethylsulfoxide (DMSO) + 20% (v/v) FCS in PB1 for 5 min and then transferred into 15.0% EG (v/v) + 15.0% DMSO (v/v) + 20% FCS + 0.5 m sucrose in PB1 (vitrification solution) for 1 min at room temperature (22–24°C). During exposure to the vitrification solution, oocytes were loaded on a Cryotop^® (Kitazato Supply Co., Tokyo, Japan). At warming, the film of Cryotop was directly immersed into PB1 containing 0.5 m sucrose and 20% FCS at 37.5°C. The warmed oocytes were washed three times and put into a HEPES-buffered (22 mm) modified R1ECM (310 mOsm) medium. The sperm heads were microinjected intracytoplasmically into the warmed oocytes. Then, presumptive zygotes were transferred surgically into the oviducts of recipient females (Day 0), and Caesarean section of the recipients was performed on Day 22. After vitrification and warming, 245 of 275 (88%) oocytes survived morphologically, 240 of the warmed oocytes were injected, and 156 oocytes (65%) were morphologically normal after the injection. To confirm development to term of zygotes derived from cryopreserved oocytes and sperm, 143 injected oocytes were transferred to 9 recipients, resulting in 3 pregnancies and the generation of one live pup. The results indicate that rat zygotes derived from cryopreserved oocytes and sperm through ICSI can develop to term, and full developmental competence can be preserved in rat oocytes after cryopreservation.