91 SPERMATOZOA TELOMERE LENGTH DETERMINES EMBRYONIC TELOMERE LENGTH BEFORE EMBRYONIC GENOME ACTIVATION

Abstract

A critical issue for species integrity is the existence of a telomere elongation program during embryogenesis that ensures sufficient telomere reserves in mammalian newborns. Two different mechanisms have been reported to act on telomere elongation during early embryogenesis: first, the telomerase, the ribonucleoprotein that adds telomeric repeats onto the chromosome ends, known to be responsible for the telomere lengthening at the morula-blastocyst transition in mice and bovine; second, in laboratory mice strains, mature oocytes increase the length of their relatively short telomeres between the 1-cell and 2-cell stages by a recombination or ALT-like pathway. In contrast, spermatozoa, the terminally differentiated male gametes, exhibit a very long telomere length (TL). The aim of this study was to clarify the potential role of the spermatozoa TL in the telomere lengthening occurring between oocyte and the 2-cell stage. For this purpose, we used 2 mouse species known to differ greatly in their TL [Mus musculus (hybrid C57CBAF1), long TL, and Mus spretus, short TL]. First, we compared relative TL in sperm samples from 5 age-matched males of each species by quantitative real-time PCR, with the numbers of telomere repeats being normalized, to the amount of DNA present in the sample (based on quantification of the Rn18S gene) by the comparative Ct method. Then, 1- and 2-cell embryos were produced by fertilizing Mus musculus oocytes with either Mus musculus or Mus spretus spermatozoa. The TL analysis in oocytes, zygotes, or 2-cell embryos was carried out by absolute quantification of telomere repeats by qPCR and normalized to the highest Ct observed value. Twenty to thirty samples per stage were analyzed, with each sample consisting in 2 matured oocytes, 2 zygotes, or one 2-cell embryo, to allow comparisons between stages. One-way ANOVA was used for statistical analysis. Mus spretus spermatozoa had significantly shorter telomeres than did Mus musculus (1.0 ± 0.1 v. 9.0 ± 1.5, respectively; P ≤ 0.01). The TL increased after fertilization from oocyte to zygote and 2-cell embryo stages in Mus musculus (1.0 ± 0.1, 1.5 ± 0.1, and 2.4 ± 0.2, respectively; P ≤ 0.01). In contrast, no differences were found in the TLs between the 3 stages in Mus spretus hybrids (oocyte: 1.0 ± 0.1; zygote: 1.0 ± 0.1; and 2-cell embryo: 1.0 ± 0.1), indicating that no elongation occurred after fertilization with spermatozoa with short telomeres. Herein, we demonstrated that before embryonic genome activation occurs, spermatozoa TL determines TL of the early embryo, suggesting that spermatozoon telomeres may act as recombination templates for early telomere lengthening right after syngamia.