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In Vitro Growth and Development of Bovine Oocyte-Granulosa Cell Complexes on the Flat Substratum: Effects of High Polyvinylpyrrolidone Concentration in Culture Medium¹

Department of Animal Production and Grasslands Farming,³ National Agricultural Research Center for Tohoku Region, Iwate 020-0198, Japan Research Institute for the Functional Peptides,⁴ Yamagata 990-0823, Japan Yamagata Agricultural Research and Training Center,⁵ Yamagata 996-0041, Japan

The aim of this study was to establish a culture system to support the growth of bovine oocytes as enclosed in granulosa cell complexes that extend on a flat substratum. Such systems have been established for mouse oocytes but are not applicable to larger animals because it is difficult to maintain an appropriate association between the oocyte and companion somatic cells. Growing bovine oocytes with a mean diameter of 95 µm were isolated from early antral follicles: the growing stage corresponds to that of oocytes in preantral follicles of 12-day-old mice. Oocyte-granulosa cell complexes were cultured for 14 days in modified TCM199 medium supplemented with 5% fetal bovine serum, 4 mM hypoxanthine, and 0.1 µg/ml estradiol. The novel modification made for this medium was a high concentration, 4% (w/v), of polyvinylpyrrolidone (PVP; molecular weight of 360 000). The flat substratum used was either an insert membrane fit in the culture plate or the bottom surface of the wells of 96-well culture plates. PVP influenced the organization of complexes, resulting in a firm association between the oocyte and the innermost layer of surrounding cells. More oocytes enclosed by a complete cell layer were recovered from the medium supplemented with 4% PVP than from the control medium. Similarly, of the oocytes initially introduced into the growth culture, a significantly larger proportion developed to the blastocyst stage from medium containing 4% PVP than from medium without PVP. When PVP medium was used, the overall yield of blastocysts was similar between the system with the insert membranes (12%) and that with the 96-well culture plates (9%). A calf was produced from one of four embryos derived from oocytes grown in 96-well culture plates, matured, and fertilized in vitro and then transferred to a recipient cow.

¹ This research was supported by a grant for the Pioneer Research Project (12072 to Y.H.) from the Ministry of Agriculture, Forestry and Fisheries of Japan and also by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences of Japan (to H.H.).

² Correspondence: Yuji Hirao, Animal Breeding and Reproduction Laboratory, Department of Animal Production and Grasslands Farming, National Agricultural Research Center for Tohoku Region, 4 Akahira, Shimo-kuriyagawa, Morioka, Iwate 020-0198, Japan. FAX: 81 19 643 3542; yujih{at}affrc.go.jp

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