Complete replication of human sperm genome in egg extracts from Xenopus laevis.
To examine the ability of Xenopus egg extracts to support a complete replication cycle of human sperm genome, demembranated human spermatozoa were incubated with the extract from activated Xenopus laevis eggs. Most sperm heads were decondensed within 15 min. The heads became round within 30 min with diameters of 10-30 microns. The process of DNA replication in the pronuclei was monitored by two methods, bromodeoxyuridine incorporation and flow cytometry. The results indicate that DNA replication was initiated approximately 1.5 h after membrane structure formation and that it lasted up to 9 h. The amounts of DNA in most pronuclei were doubled by 4-9 h, depending on which donor toad was the source of the egg extract. Inclusion of the protein synthesis inhibitor, cycloheximide (100 micrograms/ml), had no obvious effect on human sperm DNA replication but appeared to prevent the pronuclei from degradation after a prolonged period (> 6 h) of incubation. After storage in liquid nitrogen for > 1.5 mo, the efficiency of the egg extracts in supporting sperm head decondensation and DNA replication was reduced for human sperm but not for Xenopus sperm. Possible applications of the use of Xenopus egg extract for human sperm activation and DNA replication are discussed.
Xu
Y S
YS
Coriell Institute for Medical Research, Camden, New Jersey 08103, USA.
Overton
W R
WR
Marmar
J L
JL
Leonard
J C
JC
McCoy
J P
JP
Butler
G H
GH
Li
H
H
D000818:Animals; D001973:Bromodeoxyuridine / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D004261:DNA Replication / Q000187:drug effects; D005260:Female; D015894:Genome, Human; D006801:Humans; D007700:Kinetics; D008297:Male; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013045:Species Specificity; D013094:Spermatozoa / Q000378:metabolism*; D014982:Xenopus laevis
D011500:Protein Synthesis Inhibitors; D003513:Cycloheximide; D001973:Bromodeoxyuridine