Abstract
We devised a sensitive method for the site-specific detection of rare meiotic DNA strand breaks in germ cell-enriched testicular cell populations from mice that possess or lack an active recombination hot spot at the H2-Ea gene. Using germ cells from adult animals, we found an excellent correlation between the frequency of DNA breaks in the 418-bp H2-Ea hot spot and crossover activity. The temporal appearance of DNA breaks was also studied in 7- to 18-day-old mice with an active hot spot during the first waves of spermatogenesis. The number of DNA breaks detected rose as leptotene and zygotene spermatocytes populate the testis with a peak at day 14 postpartum, when leptotene, zygotene, and early pachytene spermatocytes are the most common meiotic prophase I cell types. The number of DNA breaks drops precipitously 1 day later, when middle to late pachytene spermatocytes become the dominant subtype. The recombination-related breaks in the hot spot likely reflect SPO11-induced double-strand breaks and/or recombination intermediates containing free 3′ hydroxyl groups.
We thank Mike Lichten, Tom Petes, and three anonymous reviewers for helpful comments on the manuscript. We are grateful to Peter Calabrese and Simon Tavaré for statistical advice and Cynthia Park and Debby Andreadis for caring for the animals.
This work was supported in part by NIH grants from the NIGMS (GM36745) (N.A.) and NICHD (HD31376 and HD33816) (M.A.H.).