Application of Chemically Defined Medium RD for In Vitro Maturation of Bovine Oocytes.

Abstract

Chemically defined medium would be useful for analyzing promoters or inhibitors in in vitro maturation (IVM) of bovine oocytes. IVM system of bovine oocytes, however, in chemically defined medium is not fully established. Generally, IVM of bovine oocytes is carried by use of Tissue Culture Medium 199 (TCM199) supplemented with fetal bovine serum (FBS) and gonadotropins, but sub-optimal conditions for IVM. On the other hand, Medium RD (RPMI1640 and Dulbecco's MEM, 1:1 v/v) has been shown superior to TCM199 for culture of rabbit embryos. Here we studied availability of Medium RD for IVM as a chemically defined medium of bovine cocytes. Cumulus-oocyte complexes (COCs) were aspirated from small antral follicles of ovaries obtained at a local abattoir. IVM was carried out in four different media: 10 mM HEPES buffered TCM199 supplemented with 0.1% PVP (mTCM199), mTCM199 supplemented with 20% (v/v) RD (20%RD-mTCM199), 10 mM HEPES buffered RD with 0.1% PVP (mRD), and mTCM199 supplemented with FBS fraction obtained by ultracentrifugation (mTCM199/FBS fraction). Maturation media of all groups were further supplemented with 1 µg/ml Estradiol and 0.12 IU/ml FSH. COCs were cultured for 24 h in four different maturation media. In the first experiment, we assessed cumulus expansion after IVM. Cumulus cell expansion was evaluated by measurement of cumulus diameter after 0 and 24 h of culture. The mean diameters after 24 h of culture were not different among all groups (mTCM199, 20%RD-mTCM199, mRD, and mTCM199/FBS fraction were 660 ± 36, 768 ± 47, 728 ± 35, and 710 ± 29 µm, respectively). In the second experiment, we examined the effects of different maturation media on developmental ability to the blastocyst stage after IVM. After IVM, oocytes were subjected to in vitro fertilization and subsequently cultured in the chemically defined embryo culture medium for 9 days. The rates of development to the blastocyst stage with mTCM199 (50.0%), 20%RD-mTCM199 (48.0%), mRD (55.0%), and mTCM199/FBS fraction (52.0%) were not different from one another. In the third experiment, we examined the effects of maturation media on cytoplasmic maturation after IVM. We evaluated active mitochondrial distribution of bovine IVM oocytes. Oocytes cultured in different maturation media were stained by Rhodamine 123. The stained oocytes were examined using LSM-710 confocal laser scanning microscope. In the oocytes matured with 20%RD-mTCM199, mRD, and mTCM199/FBS fraction, active mitochondria distributed uniform throughout the ooplasm. However, in the oocytes matured with mTCM199, active mitochondria seemed to be located in the peripheral than in the central cytoplasm. In summary, the present results indicate that mRD or 20%RD-mTCM199 would be useful for IVM as a chemically defined medium of bovine oocytes. (poster)