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Protamine 2 Deficiency Leads to Sperm DNA Damage and Embryo Death in Mice¹

Department of Life Science,² Kwangju Institute of Science and Technology (K-JIST), Kwangju 500-712, Republic of Korea Gamete Biology Section,³ Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 Department of Biology⁴ Center for Research on Reproduction and Women's Health and Department of Obstetrics and Gynecology,⁵ University of Pennsylvania, Philadelphia, Pennsylvania 19104

Cytokinesis is incomplete in spermatogenic cells, and the descendants of each stem cell form a clonal syncytium. As a result, a heterozygous mutation in a gene expressed postmeiotically affects all of the haploid spermatids within a syncytium. Previously, we have found that disruption of one copy of the gene for either protamine 1 (PRM1) or protamine 2 (PRM2) in the mouse results in a reduction in the amount of the respective protein, abnormal processing of PRM2, and inability of male chimeras to transmit either the mutant or wild-type allele derived from the 129-genotype embryonic stem cells to the next generation. Although it is believed that protamines are essential for compaction of the sperm nucleus and to protect the DNA from damage, this has not been proven experimentally. To test the hypothesis that failure of chimeras to transmit the 129 genotype to offspring was due to alterations in the organization and integrity of sperm DNA, we used the single-cell DNA electrophoresis (comet) assay, ultrastructural analysis, and the intracytoplasmic sperm injection (ICSI) procedure. Comet assay demonstrated a direct correlation between the fraction of sperm with haploinsufficiency of PRM2 and the frequency of sperm with damaged DNA. Ultrastructural analysis revealed reduced compaction of the chromatin. ICSI with PRM2-deficient sperm resulted in activation of most metaphase II-arrested mouse eggs, but few were able to develop to the blastocyst stage. These findings suggest that development fails because of damage to paternal DNA and that PRM2 is crucial for maintaining the integrity of sperm chromatin.

¹ This work was supported in part by grants from the Korea Ministry of Science and Technology, the Molecular Medicine Research Group Program (M1-0106-00-0007 to C.C.), the Korea Science and Engineering Foundation (1999-2-209-009-3 to C.C.), the National Institutes of Health, National Institute of Child Health and Human Development (HD22681 to R.M.S.; HD22732 to G.S.K. and R.M.S.; HD28832 to N.B.H.), and the Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health (to E.M.E.).

⁶ Correspondence: E. M. Eddy, Gamete Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709. FAX: 919 541 3800; eddy{at}niehs.nih.gov