In vitro development of bovine oocytes reconstructed with round spermatids.
The timing between round spermatid(s) (RS) injection and oocyte activation are critical for spermatid remodeling and embryo development in intracytoplasmic injection of round spermatid (ROSI) procedure. The objective of the present study was to develop an appropriate oocyte activation method for producing developmentally competent bovine embryos reconstructed with RS. Embryos reconstructed by ROSI were compared with three activation treatments for the rates of pronuclear formation, development and ploidy. RS were isolated from bull testes by Percoll density gradients. Matured oocytes were divided into three activation groups. In Group 1, oocytes were activated with ionomycin (5 microM, 5 min) before ROSI. In Group 2, oocytes were activated with ionomycin after ROSI. In Group 3, oocytes were activated twice with ionomycin before and after ROSI. All the eggs were then incubated in cycloheximide (CHX, 10 microg/mL) for 5 h and cultured in CR1aa medium for up to 8 days. Three methods of oocyte activation were also compared for the activation and development of parthenotes. Activation rates among the groups were 70-79% and did not differ. Cleavage rates in parthenotes were significantly (P < 0.05) higher in Group 3 than in Groups 1 and 2, but blastocyst rates did not differ among the groups. In ROSI embryos, the rates of cleavage and development into blastocysts were significantly (P < 0.05) greater in Group 3 (82.3% and 13.1%) than in Groups 1 and 2 (53.7, 5.8% and 64.2, 1.7%, respectively). Ploidy analysis by examining the metaphase spreads of ROSI blastocysts displayed greater numbers of diploid chromosomal complements. These results suggest that intracytoplasmic RS injection combined with repeated ionomycin activation followed by CHX treatment is more efficient for producing developmentally competent embryos.
Ock
Sun-A
SA
College of Veterinary Medicine, Gyeongsang National University, 660-701 Chinju, Republic of Korea.
Kwack
Dae-Oh
DO
Lee
Sung-Lim
SL
Cho
Sang-Rae
SR
Jeon
Byeong-Gyun
BG
Kumar
B Mohana
BM
Choe
Sang-Yong
SY
Rho
Gyu-Jin
GJ
D000818:Animals; D001755:Blastocyst / Q000502:physiology; D002417:Cattle; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D047108:Embryonic Development; D005260:Female; D015759:Ionomycin / Q000494:pharmacology; D008297:Male; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D010312:Parthenogenesis; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods / Q000662:veterinary*; D013087:Spermatids / Q000502:physiology*; D013737:Testis / Q000166:cytology; D020858:Tissue and Organ Harvesting / Q000662:veterinary
D015759:Ionomycin; D003513:Cycloheximide