380 FULL-TERM DEVELOPMENT OF MOUSE EMBRYOS PRODUCED FROM RECONSTRUCTED GIANT OOCYTES BY INTRACYTOPLASMIC SPERM HEAD INJECTION

Abstract

In mammals, unusually large diploid giant oocytes are ovulated occasionally, but as any fertilized embryos would be triploid, the potential of the cytoplasm to support normal development is unknown. However, if such giant oocytes possess normal cytoplasm, the oocytes or their excessive cytoplasm would have potential applications in the treatment of human infertility and would enhance the study of basic biology, such as the influence of cytoplasmic factors on genomic reprogramming. We reconstructed oocytes 2 to 9 times normal volume by electrofusion or mechanical fusion between intact and enucleated oocytes. First, we examined the in vitro developmental potential of 2- to 9-times giant oocytes after parthenogenetic activation. Second, 2-times giant oocytes were reconstructed by fusing with intact and enucleated oocytes; then we examined the in vitro and in vivo developmental potential of these giant oocytes after intracytoplasmic sperm injection (ICSI). Third, somatic cell nuclear transfer (SCNT) was carried out using enucleated giant oocytes. When the giant oocytes were activated parthenogenetically, most developed into morulae or blastocysts, irrespective of the original size. When sperm heads were injected into 2-times giant oocytes, these were fertilized and developed normally in vitro; after embryo transfer, we obtained 12 healthy offspring (3 female, 3 male, and 6 cannibalized the next day) by Caesarian section. All survivors grew to adulthood and demonstrated normal fertility. However, we failed to generate cloned mice by SCNT. In conclusion, reconstructed giant oocytes have normal potential for development after activation and fertilization. The reconstructed giant oocytes will provide us with interesting tools for basic biology, such as providing new material for the study of nuclear reprogramming and genomic imprinting, and may be important in widening the treatment options in human assisted reproductive technology.