Isolation and viability of presumptive spermatids collected from bull testes by Percoll density gradient.
The objective of the present study was to develop a procedure for isolating pure populations of round spermatid(s) (RS) by Percoll density gradient from bull testes. Bull testes were de-capsulated and testicular tissues were dissociated enzymatically to recover RS. After being filtered through a 20 microm nylon mesh, the cells were centrifuged at 650 x g for 25 min through the discontinuous Percoll density gradients (20, 35, 40, 45 and 90% Percoll solution). Isolated cells were analyzed by microscopic observation for survivability and apoptosis. In Experiment 1, both microscopic observation and DNA analysis by flow cytometry showed that approximately 40% of cells collected from 35% Percoll gradient were presumptive RS, whereas in 40% Percoll gradient, mostly primary spermatocytes were observed. Experiment 2 compared the effect of 35% Percoll density isolation on the incidence of apoptosis and necrosis in fresh and frozen-thawed cells to those of untreated cells. The percentage (mean+/-S.E.M.) of necrosis in cells collected from 35% Percoll gradient was less (P<0.05) than in untreated and frozen-thawed cells from 35% Percoll gradient (11.7+/-3.1% compared with 26.3+/-2.0% and 53.5+/-1.3%, respectively), but the rate of apoptosis did not differ (1.2+/-0.49% compared with 2.5+/-0.8% and 0.9+/-0.04%, respectively). The proportional data (mean+/-S.E.M.) of live cells in Percoll treated group were greater (P<0.05) than in untreated and frozen-thawed cells from the 35% Percoll gradient (86.7+/-3.26% compared with 70.8+/-2.73% and 41.9+/-1.69%, respectively). Experiment 3 compared the development rates of embryos injected with RS isolated from fresh and frozen-thawed cells collected with the 35% Percoll gradient to those of untreated cells, and parthenotes as control. There were no significant (P>0.05) differences in the rates of cleavage and blastocyst development between untreated fresh cells and fresh cells collected from the 35% Percoll gradient (75.4 and 10.5% compared with 82.4 and 12.8%). However, there were lesser (P<0.05) cleavage and blastocyst rates in frozen-thawed cells from the 35% Percoll gradient (51.6 and 6.3%) and parthenotes (60.7 and 4.1%) were observed. These results suggest that isolation of presumptive RS by 35% Percoll density gradient is effective in eliminating apoptotic and early necrotic cells. However, the use of RS in improving the developmental potential of embryos merits further studies.
Ock
Sun-A
SA
Department of Obstetrics and Theriogenology, Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Chinju 660-701, Republic of Korea.
Lee
Sung-Lim
SL
Jeon
Byeong-Gyun
BG
Cho
Sang-Rae
SR
Kumar
B Mohana
BM
Choi
Yang-Seok
YS
Choe
Sang-Yong
SY
Rho
Gyu-Jin
GJ
D000818:Animals; D017209:Apoptosis; D001755:Blastocyst / Q000166:cytology / Q000502:physiology; D002417:Cattle; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient / Q000379:methods / Q000662:veterinary*; D004247:DNA / Q000032:analysis; D047108:Embryonic Development; D005260:Female; D005434:Flow Cytometry / Q000662:veterinary; D008297:Male; D011205:Povidone; D012822:Silicon Dioxide; D013076:Sperm Count / Q000379:methods / Q000662:veterinary*; D013087:Spermatids / Q000502:physiology*; D013737:Testis / Q000166:cytology*
C016039:Percoll; D012822:Silicon Dioxide; D004247:DNA; D011205:Povidone