280 PORCINE BLASTOCYSTS DERIVED FROM IN VITRO-MATURED OOCYTES INJECTED INTRACYTOPLASMICALLY WITH SPERM FROM TESTICULAR TISSUE XENOGRAFTED INTO NUDE MICE

Abstract

The utilization of spermatogonia from testicular tissue after xenografting into immuno-deficient mice should lead to new insights for the conservation of male gametes. However, successful embryo production using sperm cells from xenografted testicular tissues has been limited to rhesus monkeys (Honaramooz et al. 2004 Biol. Reprod. 70, 1500-1503). In the present study, the objective was to establish this new technology for pig conservation in combination with intracytoplasmic sperm injection. Testes were obtained from male piglets 6 to 15 days old, in which most of the germ cells were gonocytes; these were minced into pieces of approximately 1.5 × 1.5 × 1.5 mm. Approximately 20 fragments were transplanted under the back skin of castrated nude mice 5 to 8 weeks old. The testicular grafts were recovered between 125 and 192 days after xenografting, minced in Dulbecco's phosphate-buffered saline, and centrifuged several times, to serve as a sperm suspension. In vitro maturation of the recipient oocytes (Kikuchi et al. 2002 Biol. Reprod. 66, 1033-1041) and injection with an intact spermatozoon, followed by electrical stimulation at 1 h post-injection (Nakai et al. 2003 Biol. Reprod. 68, 1003-1008), were carried out. The putative zygotes were cultured in vitro for 6 days (Kikuchi et al. 2002), and were then fixed, stained, and assessed for embryonic development and quality. From a total of 27 mice that were xenografted with testicular tissues, spermatids and spermatozoa were obtained in 19 of the mice (70.4%). Most of the spermatozoa were matured morphologically, showing faint motility after release into the collection medium. From a total of 253 oocytes (four replications) that were injected with sperm, 63 (24.9 ± 7.1%) oocytes developed to the blastocyst stage. The average total cell number was 41.9 ± 3.9. These values are comparable to those in in vitro fertilization by frozen-thawed spermatozoa, resulting in developmental ability to piglets after embryo transfer (25.3% and 48.7 cells; Kikuchi et al. 2002). These results suggest the possibility of embryo production using porcine spermatozoa that are differentiated from gonocytes within the xenografts.