95 IN VITRO MATURATION AND FERTILIZATION OF OOCYTES FROM OVARIAN TISSUES CRYOPRESERVED AND XENOGRAFTED INTO NUDE MICE

Abstract

Primordial follicles act as stores of ovarian follicles and are potential sources of oocytes for medical, agricultural, and zoological purposes. Ovarian xenografting seems to be advantageous for maturing the oocytes in primordial follicles (primordial oocytes) and useful for the conservation and reproduction of domestic or endangered animals. We have generated viable embryos from porcine primordial oocytes xenografted into nude mice (Kaneko et al. 2006 Reproduction). Xenografting of ovarian tissues after cryopreservation would be a very powerful tool for this purpose. Recently, Moniruzzaman et al. 2009 Theriogenology) reported that follicles were able to develop to the pre-antral stage in ovarian tissues after cryopreservation and xenografting, but that oocytes were not obtainable from them. In the present study, we vitrified the tissue after different immersion periods in a cryoprotectant, ethylene glycol (EG), and evaluated the possibility of oocyte collection, and also their maturation and fertilization abilities. Ovarian tissue from piglets approximately 20 days old was minced into cubes of about 1.0 to 2.0 mm. After equilibration in 4% EG in IVC-PyrLac (Kikuchi et al. 2002 Biol. Reprod.) as a base solution (BS) for 15 min, they were immersed in vitrification solution (35% EG, 5% polyvinyl pyrrolidone, and 0.3 M trehalose in BS) for 45 s or 7 min (45-s and 7-min immersion groups, respectively), then dropped with about 4 μL of vitrification solution into liquid nitrogen (LN2). After storage in LN2, microdroplets were transferred in warming solution (0.4 M trehalose in BS) at 37°C for 2 min, then consecutively transferred for 2-min periods into 0.2 M, 0.1 M, or 0.05 M trehalose in BS.As described previously (Kaneko et al. 2006 Reproduction), 20 to 30 pieces of tissue were grafted into kidney capsules of ovariectomized nude mice. The host mice were treated with porcine FSH (62.5 U mL^-1 in osmotic pomp) for 12 days before assessment of the survival of the ovarian grafts. When antral follicles were evident in the grafts, oocytes were collected using a surgical blade. They were then matured and fertilized in vitro (Kikuchi et al. 2002 Biol. Reprod.). Ovaries containing antral follicles were obtained between 62 and 125 days after grafting from 6 out of 12 mice in the both 45-s and 7-min groups. When a total of 39 and 49 fully grown oocytes, respectively, had been collected from these groups and cultured, the maturation rates calculated on the basis of 1st polar body extrusion were 18% (7/39) and 33% (16/49), respectively. The corresponding rates for sperm-penetrated oocytes were 83% (5/6) and 88% (14/16), respectively. All the oocytes formed male and female pronuclei at 10 h after insemination. The rates in the 2 immersion groups did not differ significantly by chi-square test, with or without Yates’ correction. In conclusion, fully grown porcine oocytes can be collected from primordial follicles that have been cryopreserved and xenografted into nude mice. The period of immersion before vitrification may not affect oocyte maturation or fertilization ability.