Early preantral mouse follicle in vitro maturation: oocyte growth, meiotic maturation and granulosa-cell proliferation.

摘要

Mechanically isolated early preantral mouse follicles were cultured singly for 16 days. The number of granulosa cells per follicle was determined at 2-day intervals using a Neubauer-counting chamber. The diameter and meiotic status of oocytes were determined under an inverted microscope. Proliferation rate was slow when the granulosa cells were within the basal membrane. From day 6, when granulosa cells had broken through the basal membrane, proliferation rate increased up to day 10 and decreased thereafter to approximately 12 000 cells per culture droplet. Incorporation of BrdU revealed that proliferating cells were evenly distributed throughout the follicle until antrum formation. As granulosa cell differentiation progressed, proliferation of mural granulosa cells ceased, while cells around the oocytes continued dividing. Oocyte diameter increased discontinuously in relation to follicle remodelling. During the 1st growth phase, oocyte diameter increased from 56.5±4.4μm to 67±4.1μm until the onset of antral-like cavity formation. The last growth phase started after day 10, and by day 14 oocyte diameter was not significantly different from that of 26-day-old in vivo-grown control oocytes. The potential to resume meiosis after mechanical removal of granulosa cells was first reached on day 8; thereafter, removal of the corona showed that all oocytes cultured with FSH remained arrested at the germinal vesicle (GV) stage up to day 16. After day 8, approximately 70% of all oocytes had undergone GVbreakdown (GVBD) as a result of granulosa-cell removal, but only 23% of these had reached the MII stage after 24 h. Control oocytes showed 60% GVBD when the granulosa was removed, but most of the oocytes (82%) underwent 1st polar body extrusion 24 h later. The results suggest that although oocyte diameter after in vitro maturation is not different from that of in vivo matured oocytes, culture conditions are not adequate to support complete meiotic maturation.