| Co-Investigator(Kenkyū-buntansha) |
MIYANO Takashi Kobe University, Faculty of Agriculture, Associate Professor, 農学部, 助教授 (80200195)
MIYAKE Masashi Kobe University, Graduate School of Science and Technology, Professor, 大学院・自然科学研究科, 教授 (60093316)
KANNAN Yasuyuki Kobe University, Faculty of Agriculture, Professor, 農学部, 教授 (60031192)
TOMINAGA Keiichiro Hyogo Prefectural Agricultural Institute, Senior Investigator, 主任研究員
HARAYAMA Hiroshi Kobe University, Graduate School of Science and Technology, Associate Professor, 大学院・自然科学研究科, 助教授 (30281140)
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| Research Abstract |
Several hundred thausand primordial follicles are contained in the ovaries of domestic animals. The development of in vitro culture systems for the growth of small oocytes in primordial or small follicles is expected to provide a new source of a large population of oocytes for livestock production. This study was conducted to develop Culture systems for small oocytes from pig ovaries.
Preantral follicles containing oocytes with various sizes were isolated from pig ovaries and cultured in collagen gel for up to 16 days in the presence of serum, FSH and oestradiol. Waymouth MB7 52/1 was used as a basic culture medium for oocyte growth. After culture, the oocytes enclosed by one or two layers of cumulus cells were cultured for meiotic maturation and inseminated with spermatozoa. The results obtained were as follows.
1. Pig growing oocytes grew to their final size, acquired meiotic competence du ring in vitro culture, and were penetrated by spcermatozoa in vitro, although there was a very lo
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w level of success in fertilization. In contrast to the growing oocytes, no culture methods were available for much smaller oocytes. We tried to culture primordial follicles which contained small oocytes 30 μm in diameter, but the integrity of the follicles was soon broken and the follicles survived for only a few days.
2. Using culture systems, we demonstrated that granulosa cells regulate oocyte growth, and that antrum formation of granulosa cells was induced only by meiotic-competent oocytes, indicating that the oocytes secrete a substance(s) that induces differentiation of granulosa cells.
3. Of four glycosaminoglycans examined, hyalronic acids and chondroitin sulfate A supported the development of in vitro-matured and -fertilized pig oocytes to the blastocyst stage.
4. In addition, degradation of cyclin B1 molecules and MAP kinase deph osphorylation during fertilization, localisation of phosphorylated MAP kinase during the transition from meiosis I to meiosis II, assembly and transformation of the spindle during the progression through meiotic cell cycle, and the level and localization of CENP-E and kinetochore numaber during maturation were examined. Less
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