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A Reproducible Two-Step Culture System for Isolated Primary Mouse Ovarian Follicles as Single Functional Units¹

Follicle Biology Laboratory, Vrije Universiteit Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium

A reproducible two-step culture system for isolated mouse ovarian follicles smaller than 100 µm (type 3a follicles) was designed. First, isolated follicles were grown in single droplets of -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 µm were transferred to -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 µm. 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 ß deficiencies during in vitro culture.

¹ Supported by the Institute for the Promotion of Innovation by Science and Technology in Flanders (STWW 980343).

² Correspondence: Sandy Lenie, Follicle Biology Laboratory, Vrije Universiteit Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium. FAX: 32 02 477 5060; sandy.lenie{at}vub.ac.be

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