In vitro production of viable eggs from isolated mouse primary follicles by successive culture

To produce viable eggs from single primary follicles in vitro, primary follicles containing oocytes (average 39.0 ± 0.2 µm in diameter) were isolated from the ovaries of 1-week-old mice, and cultured in combination with culture membranes for the first 8 days up to the secondary follicle stage, followed by the next 12 days to the later stages. After culture with a combination of first and second culture membranes using high and low adhesion characteristics, the average oocyte diameters of the surviving follicles increased by almost two-fold in all four groups. Further, the oocyte maturation rate was the highest (74.1%) in the culture group with low adhesion with collagenase and high adhesion. In this culture group, when the O(2) concentration was changed from 20% in the first culture to 5% in the second culture, the cleavage rate increased to 47.5%, which was comparable to the level of the in vivo control (34.6%). Finally, 39 embryos at the 2- to 8-cell stages were transferred into the oviducts of three pseudopregnant females, and eight live pups (20.5%) were obtained. Of the eight pups, six survived for at least six months and were fertile. The present study shows successive in vitro cultures of single isolated primary follicles for the production of viable eggs. We believe that this culture system, with a combination of culture membranes under controlled O(2) conditions, is applicable to other mammalian species, including humans.