DNA topoisomerase II enzyme activity appears in mouse sperm heads after fertilization
Sperm suspensions of 4 male mice (A, B, C and D), having an initial motility grade of 3.5 were used to examine the presence of DNA topoisomerase II (top 2) activity in sperm heads. The initial percentage motile of male A was 75%, male B was 80%, male C was 70% and male D was 60%. Top 2 activity was examined by testing the effect of etoposide (ETO), a specific top 2 blocker, on sperm motility, fertilizing ability and formation of the male pronuclei. Sperm suspension drops (0.5 ml) and fertilization drops (0.4 ml) were made from TYH medium with 50 μg/ ml ETO (treatments) or TYH without ETO (controls). Sperm suspensions were made from epididymal sperms of the above males in treatments and controls and incubated for 3 h. Mature mouse oocytes (n = 461) were co-cultured with capacitated sperms in the treatments fertilization drops for 5 h. Other oocytes (n= 437) were co-cultured with capacitated sperms in the controls fertilization drops. The oocytes were further cultured for 24 h in KSOM with ETO (for treatments) and KSOM without ETO (for controls). Six sperm motility indexes (SMI) for each male were recorded at 30 min interval according to the formula, SMI = (grade)2 × % motile. The fertilization rates and nuclear events were assessed by observing the pronuclei under an inverted microscope and finding the sperm heads in whole mounts from the oocytes that failed to make pronuclei. The SMI of the treatments and the controls increased gradually and reached peak values after 2 h of incubation. No differences (p > 0.05) were observed among SMI of the treatments and the controls. However, treatment reduced (p < 0.05) the fertilization rate and completely inhibited the formation of the pronuclei. All the oocytes fertilized in treatments (n = 412) failed to form pronuclei and all had enlarged or decondensed sperm heads, whereas 92.6% of the oocytes fertilized in the controls (n= 378) had pronuclei and only 30 (7.4%) oocytes had enlarged or decondensed sperm heads. Neither sperm motility nor fertilization were inhibited with ETO. However, the formation of the pronuclei was blocked. It was concluded that ETO has no effect on sperm capacitation and top 2 activity appears in mouse sperm heads after oocyte penetration.
Key words: DNA topoisomerase II, sperm, treatment, mice.