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Abstract. To develop a reliable follicle culture system, mouse preantral follicles 150-200 µm in diameter were cultured individually for 5 or 6 days in membrane inserts or in droplets, and then induced to ovulate with hCG (Experiment 1). The nuclear maturation and developmental competence of the oocytes that ovulated from the follicles cultured in inserts were determined (Experiment 2). There was no significant difference between the two culture systems in the survival rate (83 and 77%). However, follicles cultured in inserts showed a higher ovulation rate (63%) than those cultured in droplets (39%, P<0.05). About 80% of the oocytes that ovulated from the follicles cultured in inserts were at the metaphase II stage. After in vitro fertilization, 75 and 48% of in vitro ovulated oocytes cleaved and developed into blastocysts, respectively. These results demonstrate that the insert culture system is superior to the droplet culture system in terms of follicular growth and ovulation, and can be used to investigate the growth and ovulation of follicles in vitro. Key words: Culture system, In vitro ovulation, Mouse preantral follicle, Oocyte maturation (J. Reprod. Dev. 50: [579][580][581][582][583][584][585][586] 2004) he culture of preantral follicles provides the means not only of producing a uniform population of developmentally competent oocytes but also of investigating the physiology of follicular development and ovulation in the absence of systemic influences. Different culture systems have been developed for mouse preantral follicles [1][2][3]. Some culture syste ms maintain the threedimensional structure of follicles, while others allow adherence to an underlying substrate causing the collapse of the follicle, resulting in a twod i m e n s i o n a l s t r u c t u r e . T h e c u l t u r e o f enzymatically isolated preantral follicles produces many fertile oocytes, but the enzymatic treatment removes the thecal cells and perforates the basal membrane so that an antrum will not form [4]. A culture system that allows the attachment of follicles yields a large number of oocytes but can not retain the follicle structure [5]. A threedimensional culture has many advantages over a two-dimensional culture for investigating the mechanism of folliculogenesis, oocyte growth, ovulation and ovarian dysfunctions. Maintenance of the three-dimensional structure of isolated intact follicles has been achieved by various approaches such as culturing in membrane inserts [6], or simply by the daily transfer of follicles to new wells [7], or new droplets [8]. However, the efficiencies of these three-dimensional culture systems have not been evaluated by direct comparison under the same culture conditions. Moreover, only a few

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