Abstract
To compare the efficiency of three culture media in preantral follicle maturation. Culturing preantral follicles (PFs) by hanging drop method (HDM) at early stage of culture may ease spatial restriction, prevent premature ovulation and synchronize development. At later stage, the medium is optimized to facilitate meiosis. PFs from 15-day old ovaries were allocated into groups A,B and C. 3-5 follicles were pooled into a 25μl medium droplet and cultured for 6 days by HDM. Aggregates were transferred to 30ul medium droplets covered with oil at day 7. Media used are: A, Medicult +100IU/l FSH for 12 days, followed by 1.5IU/ml hCG on day13. B, SAGE+100IU/l FSH for 12 days, followed by hCG. C, α-MEM+ 10% FBS + ½ ITS + 100IU/l FSH (MIF) for 10 days. Additional 25μM Vitamin C and 2μM RA were added at day11. The medium was further added with 10ng/ml EGF and 1.5IU/ml hCG at day13. Prior to culture in CytoD, MII oocytes were treated with 5μM ionomycin for 20 min. Blastocyst formation of activated ooctyes was scored. Table 1Comparison of different media in oocyte maturation from preantral follicles.Experimental group<12-day culture<12-day culture<12-day culture<48hr after hCG (total)<48hr after hCG (total)<48hr after hCG (total)MediumFollicles collectedGVGVBDMature (%)GVGVBDMature (%)A (Medicult)922810018150 (0)B (SAGE)1023838127423 (3)C (MIF)1013252784333 (33)* the percentage was calculated based on the total number of follicles collected; Numbers of oocytes not listed in the talbe are those degenerated. Open table in a new tab * the percentage was calculated based on the total number of follicles collected; Numbers of oocytes not listed in the talbe are those degenerated. Although optimized for human oocyte IVM, both Medicult and SAGE systems failed to produce mature mouse oocytes due to premature rupture. MIF is able to sustain expansion, ovulation, and meiotic maturation of the COCs with a 33% success rate. ∼20% of the MII oocytes developed into blastocysts by parthenogenesis, indicating a good ooctye competency. Our IVM system is effective to ensure follicle/oocyte maturation from early preantral stage.
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