Paternal Pronuclei in Mouse Zygotes Created by Injection with Sperm with Fragmented DNA Have Delayed and Incomplete DNA Replication.

Abstract

We previously reported that when vas deferens sperm are treated with Mn+2 and Ca+2 they cleave their DNA to loop-sized fragments of 25 to 50 kb in a process termed Sperm Chromatin Fragmentation (SCF). SCF mirrors some aspects of somatic cell apoptosis in that the DNA degradation is mediated by reversible double strand breaks caused by topoisomerase 2B (TOP2B), followed by irreversible DNA degradation by a nuclease(s). We also reported that when vas deferens SCF spermatozoa were injected into oocytes, the resulting paternal pronuclei did not initiate DNA replication, even though the maternal pronuclei replicated normally. Furthermore, the paternal DNA appeared to be degraded as evidenced by ethidium bromide staining. Here, we followed the fate of the paternal DNA in zygotes created by injection of sperm that had been induced to fragment their DNA (SCF-zygotes). We first tested whether the TUNEL assay could detect the DNA fragmentation in paternal pronuclei of SCF-zygotes. We found that even though we could verify that the sperm DNA had been fragmented to about 25 kb by field-inversion gel electrophoresis, the TUNEL assay was not sensitive enough to identify these breaks. We next tested for H2AFX phosphorylation to determine whether SCF-zygotes recognized the DNA damage. We found that paternal pronuclei resulting from injecting oocytes with SCF spermatozoa stained positively for phoshorylated H2AX at 5 hrs after fertilization, just before DNA synthesis, while the maternal pronuclei were negative. In control embryos injected with untreated sperm, neither pronuclei were positive. We then tested our original assertion that paternal pronuclei in SCF-zygotes did not replicate their DNA. In our previous study, we examined embryos at 7 hrs after fertilization, about 2 hrs after DNA synthesis normally initiates, and found that very few paternal pronuclei in SCF-zygotes initiated DNA synthesis. Here, we examined the zygotes at several time points up to 20 hrs after fertilization. We found that SCF-zygotes created after injection of vas deferens sperm with fragmented DNA did replicate paternal DNA, but that the initiation was delayed for up to 10 hrs. Chromosomal analysis of these zygotes demonstrated that in the majority of them paternal chromosomes were abnormal and prematurely condensed, while the maternal pronuclei formed normal chromosomes. A significant number of chromosome breaks were also detected in the paternal complements. These data demonstrate that even though the one-cell embryo recognizes damaged DNA in paternal pronucleus, it still attempts to replicate it, although the replication is much delayed, and may not be complete. This delay may be an attempt by the embryo to repair the DNA breaks before replication is initiated. If so, the premature condensation of paternal chromosomes (PCC) may be the result of the asynchronous initiation of DNA synthesis in the two pronuclei; completed DNA synthesis and initiation of mitosis in the maternal pronucleus causing the incompletely replicated paternal DNA to condense prematurely. Alternatively, the paternal PCC may represent damaged DNA that could not be replicated. In either case, the data demonstrate that sperm DNA damage that is below the level detectable by TUNEL can severely affect early embryo development. This research was supported by NIH grant HD060722.