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Effect of Cycloheximide on Bovine Oocyte Nuclear Progression and Sperm Head Transformation after Fertilization In Vitro

  • Liu, L. (Laboratory of Reproductive Endocrinology & Embryo Engineering in Domestic Animals, Northwest Agricultural University) ;
  • Zhang, H.W. (Laboratory of Developmental Biology, Shandong University) ;
  • Qian, J.F. (Laboratory of Reproductive Endocrinology & Embryo Engineering in Domestic Animals, Northwest Agricultural University) ;
  • Fujihara, N. (Department of Animal Reproduction, College of Agriculture, Kyushu University)
  • Received : 1998.02.28
  • Accepted : 1998.07.14
  • Published : 1999.01.01

Abstract

Bovine oocytes with compact and complete cumulus cells were cultured in 6 groups for up to 24h in TCM199 buffered with 25 mmol/1 HEPES and supplemented with 10% FCS, 1 mg/ml $17{\beta}$-estradiol, 20 IU/ml hCG. Half of the oocytes at each group cultured in the presence of $25{\mu}g/ml$ cycloheximide at different times during maturation (0, 6, 12, 18, 20, 22 h) were fixed at 24 h of maturation to examine the nuclear progression. The rests of them were inseminated with frozen-thawed spermatozoa in medium BO with 10 mg/ml BSA and 10 mg/ml heparin and fixed after additional 18-20 h culture to evaluate the sperm head transformation. When a protein synthesis inhibitor was added at the onset of the maturation, the oocytes were prevented to proceed GVBD. A few of the oocytes (16%) were able to be penetrated and sperm head decondensation was inhibited either. Addition of cycloheximide after 6-12 h of culture resulted in an increasing percentage of GVBCD (more than 80%), but the oocytes became arrested in M-I (69.2%). More than half of the oocytes was penetrated with a decondensing sperm head. Formation of male pronucleus was first obtained at 12 h of culture in the presence of cycloheximide. When cycloheximide was added from 18 h of culture onwards, nuclear progression to M-II was increasingly restored (80.4-85.5%). The proportion of male and female pronuclear formation increased from 17.9% to 46.2%. It is concluded that protein synthesis is necessary not only for GVBD and development from M-I to M-II, but also for sperm head decendensation and male pronuclear formation in bovine oocytes.

Keywords

Protein Synthesis;Bovine Oocytes;Maturation;Sperm Nucleus

Acknowledgement

Supported by : GOHO Life Science Foundation