Effect of preantral mouse follicle culture period on meiotic maturation and developmental competence of oocytes
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ORIGINAL ARTICLE
Effect of preantral mouse follicle culture period on meiotic maturation and developmental competence of oocytes Shouko Nonowaki • Katsuhiko Takahashi Toshitaka Horiuchi
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Received: 13 October 2009 / Accepted: 26 November 2009 / Published online: 23 December 2009 Ó Japan Society for Reproductive Medicine 2009
Abstract Purpose The aim of this study is to determine the optimal culture period for meiotic maturation and developmental competence of in vitro-grown mouse oocytes. Methods Early preantral follicles with diameter of 100– 130 lm were collected mechanically from day 14 mouse ovaries and cultured for 8, 10, and 12 days. The diameters of follicles and oocytes were measured, and chromatin configuration in oocytes was observed. We also examined meiotic maturation by human chorionic gonadotropin (hCG)/epidermal growth factor (EGF) stimulation, developmental competence of fertilized oocytes to blastocysts, and apoptosis in blastocysts. Results The follicular diameter increased significantly from days 4 to 10, and the diameter of day 12 oocytes was significantly larger than day 8 or earlier oocytes. Chromatin configuration around the nucleolus was transformed from ‘‘nonsurrounded (immature)’’ to ‘‘surrounded (mature)’’ after 10 days. Furthermore, MII rate of day 10 and 12 oocytes was significantly higher than that of day 8 oocytes. The blastocyst rate of day 10 oocytes was higher than that of day 8 or 12 oocytes. The blastocyst apoptotic rate of day 12 oocytes was higher than that of day 10 oocytes. Conclusions Long culture periods of in vitro-grown oocytes affect meiotic maturation, developmental competence to blastocysts, and apoptosis.
S. Nonowaki T. Horiuchi (&) Graduate School of Comprehensive Scientific Research, Prefectural University of Hiroshima, Hiroshima 727-0023, Japan e-mail: [email protected] S. Nonowaki K. Takahashi Hiroshima HART Clinic, Hiroshima 730-0051, Japan
Keywords Apoptosis Culture period In vitro growth Mouse Preantral follicle
Introduction Mammalian ovaries contain many follicular oocytes; however, only very few go on to develop into mature oocytes in vivo. Using in vitro culture techniques that encourage follicular growth and oocyte maturation, it is possible to accelerate the maturation of large numbers of viable oocytes. To date, the two in vitro culture systems used most often to grow preantral mouse follicles are mechanical dissection of follicles that are then individually cultured in microdroplets [1], and enzymatic isolation of oocyte–granulosa cell complexes that are then cultured on a collagen-coated membrane [2]. Mechanical dissection is useful for studying the mechanism of follicular development in the presence of thecal cells, but it cannot be performed on a large scale [1]. Furthermore, there is the risk that follicles may be affected by steroid hormone outflow into the oil covering. In contrast, although enzymatic isolation of follicles results in the loss of thecal cells and the original follicular form, with this procedure, large numbe
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