Relationship between DNA fragmentation and nuclear status of in vitro-matured porcine oocytes: role of cumulus cells
Pimprapar Wongsrikeao A , Takeshige Otoi A C , Masako Murakami A , Ni Wayan Kurniani Karja A , Agung Budiyanto A , Masao Murakami A , Masaru Nii B and Tatsuyuki Suzuki AA Laboratory of Animal Reproduction, United Graduate School of Veterinary Sciences, Yamaguchi University, Yamaguchi 753-8515, Japan.
B Tokushima Prefectural Livestock Experiment Station, Anan, Tokushima 774-0047, Japan.
C Corresponding author. Email: otoi@yamaguchi-u.ac.jp
Reproduction, Fertility and Development 16(8) 773-780 https://doi.org/10.1071/RD03099
Submitted: 22 October 2003 Accepted: 31 October 2004 Published: 13 January 2005
Abstract
The present study was conducted to investigate the effects of the attachment of cumulus cells to oocytes and coculture with cumulus cells during maturation culture on the nuclear status and DNA fragmentation of porcine denuded oocytes (DOs). In the first experiment, cumulus cells were removed from cumulus–oocyte complexes (COCs) at 0, 8, 16, 24 or 32 h after the onset of maturation culture and the DOs were then cultured in their original droplets until 42 h of culture was reached. In the second experiment, all COCs were denuded before the onset of culture and the DOs were cocultured with their removed cumulus cells. The DOs were transferred into fresh medium at 0, 8, 16, 24 or 32 h after the onset of coculture with cumulus cells and then cultured until 42 h of culture was reached. After culture, DNA fragmentation and the nuclear status of oocytes were examined using the terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling (TUNEL) method. When the DOs were returned to the same droplets after removal of the cumulus cells, the removal of the cumulus cells after 16 h of culture significantly decreased the proportion of oocytes remaining at the germinal vesicle (GV) stage. However, coculture treatment of DOs in the presence of their removed cumulus cells had no significant effects on the GV breakdown (GVBD) of oocytes. There were no significant differences in the proportion maturing to MII oocytes among the groups following removal of cumulus cells after the onset of maturation culture; however, DOs cocultured with cumulus cells until the end of maturation culture exhibited an increased maturation rate compared with DOs cocultured for 8 and 16 h. The total proportion of TUNEL-positive oocytes of oocytes remaining at the GV stage was higher than that of oocytes reaching other stages, irrespective of the removal of cumulus cells and coculture treatments. However, coculture for more than 16 h decreased the total proportion of TUNEL-positive oocytes. Our results indicate that the attachment of cumulus cells to oocytes may have a critical role for oocytes undergoing GVBD and that coculture with cumulus cells promotes the ability of oocytes to complete maturation. Moreover, coculture with cumulus cells may assist the oocyte to avoid undergoing DNA fragmentation.
Extra keywords: : apoptosis, meiotic competence.
Acknowledgment
The authors thank the staff of the Meat Inspection Office of Kitakyushu city, Japan, for supplying us with pig ovaries. We also thank Mr Kenneth Bruhn for revising the English version of the manuscript.
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