Abstract
ABSTRACTMammalian sperm are carriers of not only the paternal genome, but also the paternal epigenome in the forms of DNA methylation, retained histones and noncoding RNAs. Although paternal DNA methylation and histone retention sites have been correlated with protein-coding genes that are critical for preimplantation embryonic development, physiological evidence of an essential role of these epigenetic marks in fertilization and early development remains lacking. Two miRNA clusters consisting of five miRNAs (miR-34b/c and miR-449a/b/c) are present in sperm, but absent in oocytes, and miR-34c has been reported to be essential for the first cleavage division in vitro. Here, we show that both miR-34b/c- and miR-449-null male mice displayed normal fertility, and that intracytoplasmic injection of either miR-34b/c- or miR-449-null sperm led to normal fertilization, normal preimplantation development and normal birth rate. However, miR-34b/c and miR-449 double knockout (miR-dKO) males were infertile due to severe spermatogenic disruptions and oligo-astheno-teratozoospermia. Injection of miR-dKO sperm into wild-type oocytes led to a block at the two-pronucleus to zygote transition, whereas normal preimplantation development and healthy pups were obtained through injection of miR-dKO round spermatids. Our data demonstrate that miR-34b/c and miR-449a/b/c are essential for normal spermatogenesis and male fertility, but their presence in sperm is dispensable for fertilization and preimplantation development.
Highlights
Once inside the oocyte, the sperm head, with the paternal genome heavily packed inside, starts to de-condense and forms the paternal pronucleus (Yanagimachi, 2003; Yanagimachi, 2005a; Yanagimachi, 2012)
Some sperm DNA methylation patterns appear to be preserved after post-fertilization reprograming, and loci associated with retained histones have been correlated with genes critical for early development (Hammoud et al, 2009; Brykczynska et al, 2010; Smith et al, 2012)
Intracytoplasmic sperm injection (ICSI), and round spermatid injection (ROSI), we report, here, that these five miRNAs are expressed in sperm and absent in oocytes, they are dispensable for fertilization, oocyte activation, first cleavage division and all subsequent steps of preimplantation development
Summary
The sperm head, with the paternal genome heavily packed inside, starts to de-condense and forms the paternal pronucleus (Yanagimachi, 2003; Yanagimachi, 2005a; Yanagimachi, 2012). The developmental rates at the 2-PN and 2-cell stages remained significantly lower in the miR-dKO group, as compared to the WT control group (Table 1) These results suggest that miR-dKO sperm neither activate WT oocytes nor support the first cleavage division and the subsequent early embryonic development. MiR-dKO round spermatids can fertilize WT oocytes and support embryonic development Given the severe spermiogenic defects in miR-dKO males, the inability of miR-dKO sperm to fertilize WT oocytes and support early development may well result from structural abnormalities due to aberrant spermiogenesis, which could include the lack of the five miRNAs, and the absence of other essential factors or compromised integrity of the paternal genome. out of the dysregulated late spermiogenesis-essential genes were confirmed using qPCR analyses (Fig. 5E) Disruptions of these genes should be, at least in part, responsible for the defective chromatin condensation and flagellar formation observed in the dKO male mice. These molecular analyses suggest that ablation of these five miRNAs causes transcriptomic changes due to the dysregulation of both target and non-target genes
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