A reproducible two-step culture system for isolated primary mouse ovarian follicles as single functional units

A reproducible two-step culture system for isolated mouse ovarian follicles smaller than 100 microm (type 3a follicles) was designed. First, isolated follicles were grown in single droplets of alpha-minimal essential medium (MEM) without (deoxy)ribonucleosides at a lower concentration of fetal bovine serum (FBS; 1%) for 6 days with mechanical prohibition of thecal cell attachment. Growing follicles reaching at least 100 microm were transferred to alpha-MEM medium enriched with a higher concentration (5%) of FBS to allow attachment and were cultured subsequently for an additional 12 days. Overall, more than 85% of the follicles survived the first culture step, and oocyte growth and granulosa cell proliferation had increased by 25% (P < 0.05). Follicle survival at Day 18 was related to initial follicle diameters at isolation. Average meiotic maturation rates and estrogen secretion were lower compared to those of cultures starting with early preantral follicles of 100-130 microm. Although reverse transcription-polymerase chain reaction analysis revealed the presence of LH-receptor mRNA in thecal cells, an exogenous androstenedione replacement resulted in an increase of estrogen production, suggesting substrate insufficiency. The time needed to grow from early preantral stages to in vitro ovulation is strongly dependent on the initial follicle diameter at isolation. Morphological characteristics of cultured follicles were suggestive for combined transforming growth factor beta deficiencies during in vitro culture.