Capacity of Mouse Oocytes from Preantral Follicles to Undergo Embryogenesis and Development to Live Young after Growth, Maturation, and Fertilization in Vitro1

Abstract

A system is described here by which live mice can be produced from oocytes isolated from 12-day-old mice, be grown, matured, and fertilized in vitro, and then be transferred to pseudopregnant females. These oocytes were, at the time of isolation from preantral follicles, in about mid-growth phase and incompetent of undergoing germinal vesicle breakdown (GVB) without further development. The developmental competence of mouse oocytes that grew and underwent maturation in vitro was compared to oocytes that grew in vivo and underwent maturation in vitro. After isolation from mice 16 through 28 days old, oocytes were found to increase in size and to sequentially acquire the ability to undergo GVB, produce a polar body, cleave to the 2-cell stage after insemination, and develop to the blastocyst stage. Moreover, the number of cells per blastocyst increased with the age of the mice from which the immature oocytes were isolated. Oocyte-granulosa cell complexes isolated from 12-day-old mice were cultured for 10 days. At the end of the culture period, the oocytes had grown to a size equivalent to oocytes isolated from 16-day-old mice, and 87% of the in-vitro-grown (IVG) oocytes underwent GVB; 79% of these produced a clearly visible polar body when maturation occurred in the presence of follicle-stimulating hormone (FSH). The IVG oocytes cleaved to the 2-cell stage after insemination in vitro with a frequency equivalent to superovulated ova and ova that matured in vitro after isolation from 22-day-old mice. Twenty-four percent of the 2-cell embryos derived from IVG oocytes developed to the expanded blastocyst stage. This frequency was equivalent to that by 2-cell embryos derived from oocytes isolated from 18-day-old mice. The blastocysts derived from IVG oocytes contained the same number of cells as the blastocysts derived from oocytes isolated from 18- and 20-day-old mice. Live young were produced by the transfer of 2- or 4-cell embryos derived from IVG oocytes, but at a low frequency (5.1%). These results demonstrate for the first time that oocytes from preantral follicles of mice can complete growth and acquire full developmental competence in vitro so that live young can be produced after maturation and fertilization in vitro and transfer to foster mothers.