Experimental design of embryonic diapause (ED) induction in ovine blastocysts by transfer into ovariectomised pseudo-pregnant mice at 2.5 dpc.

<p>Following uterine flushing, diapausing ovine blastocysts were analyzed or transferred to foster ewes at day 6 after oestrus for full term development. The timing indicated in the diagram refers to embryos.</p

Reversibility of growth arrest in ovine embryos following flushing from the uterus of ovariectomised mice

<p>. (<i>a</i>) Ovine blastocysts before (i) and immediately after (ii) transfer to mouse uteri, and following 12 hours of culture in vitro (iii). (<i>b</i>) Percentage of BrdU-positive, proliferating cells and of embryos hatching from the <i>zona pellucida</i> in diapaused ovine blastocysts after 48 hours in culture and number of offspring developed from diapaused ovine blastocysts following their transfer into receptive uteri of foster ewes. Controls were in vitro cultured ovine blastocysts (day 6.5). For each experiment ≥5 blastocysts were used and it was repeated 3–5 times. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033027#s2" target="_blank">Results</a> are mean ± S.E. M *p<0.05.</p

Confirmation of ED in ovine embryos.

<p>(<i>a</i>) Proportion of BrdU- and TUNEL-positive cells in diapausing ovine and murine blastocysts flushed from ovariectomised mouse uteri and in controls (<i>b</i>) qRT-PCR analysis of genes involved in ED control. Genes that positively regulate cell proliferation (<i>PCNA</i>) and signaling (<i>HB-EGF</i>) were not expressed in diapausing ovine blastocysts, while the anti-proliferative gene <i>BTG1</i> was significantly over-expressed. <i>IGF2R</i> mRNA expression did not differ statistically between diapausing and control blastocysts. (c) Immunolocalization of CB1 (green) in diapausing (middle panel) and control ovine blastocysts (upper panel). Nuclei (red) were visualized with propidium iodide. CB1 expression is higher in diapausing ovine blastocysts. Lower panel: ovine blastocysts incubated with neutralized anti-CB1 antibody showing no positive signal. For each experiment ≥5 blastocysts were used and it was repeated 3–5 times. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033027#s2" target="_blank">Results</a> are mean ± S.E.M. *** p<0.0001, ** p<0.003, *p<0.03.</p

Induction of γH2AX in pronuclei of oocytes injected with nuclei, either from fresh, frozen or freeze-dried lymphocytes.

<p>(a) PI = pronuclei stained with propidium iodide; γH2AX = phosphorylated H2AX, a marker of DSBs and, indirectly, of DNA repair; Merge = PI+γH2AX. (b) The γH2AX fluorescent intensity was higher in pronuclei of SCNT-derived embryos using nuclei from freeze-dried cells than in pronuclei of embryos obtained using nuclei from frozen (p<0.0001) or fresh cells (p<0.0001); (Kruskal-Wallis test). Data represent the mean ± SEM of 10 embryos for each group. Scale bar = 10 µm.</p

Occurrence of DNA damage in frozen and freeze-dried lymphocytes.

<p>(a) DNA breakage in fresh vs frozen lymphocytes and (b) in fresh vs freeze-dried lymphocytes. Left profile: red line = fresh (control) cell counts; green line = frozen lymphocytes cell counts; blue line = proportion of cells with damaged DNA. Right profile: red line = fresh (control) cell counts; green line = freeze-dried lymphocytes, cell counts; blue line; proportion of cells with damaged DNA. (c) Percentage of cells with DNA fragmentation (8.2% frozen and 39.2% freeze-dried cells with DNA damage) (p<0.0001, Fisher's exact test). DNA break of X-axis is the alpha t index.</p

Ultrastructural appearance of fresh (a), frozen (b) and freeze-dried lymphocytes (c).

<p>N, nucleus; M, mitochondria, ER, eterochromatin; black arrow, intact nuclear membrane; blue arrow, intact cell membrane; red arrow, broken cell membrane; green arrow, damaged mitochondria Bar = 1000 nm.</p

In vitro development of enucleated oocytes injected with nuclei from freeze-dried and fresh cells.

<p>Embryos were cultured for 7 days. Reconstructed embryos were checked at day 1, 5, 6 and 7 for development;</p>a<p>p = 0.0035;</p>b<p>p = 0.001;</p>c<p>p = 0.0024 (χ² test).</p

γH2AX immunostaining in pronuclei of oocytes injected with up to four nuclei from freeze-dried lymphocytes.

<p>NT = nuclear transfer, 4NT = 4 pronuclei (one fragmentated), PI  = pronuclei stained with propidium iodide; γH2AX = phosphorylated H2AX, a marker of DSBs and, indirectly, of DNA repair; Merge = PI+γH2AX. Scale bar = 20 µm.</p

Metaphase spreads of SCNT-derived, 2–4 cell stage embryos.

<p>Normal metaphase spreads with 54 chromosomes from embryos obtained by injection of nuclei from (a) fresh, (c) frozen and (e) freeze-dried lymphocytes. Abnormal metaphase spreads from embryos obtained by injection of nuclei from (b) fresh (structural chromosomal abnormalities and chromosome breaks), (d) frozen (55 chromosomes) and (e) freeze-dried lymphocytes (structural chromosomal abnormalities and chromosome breaks).</p

Blastocysts produced by nuclear transfer of fresh and freeze-dried lymphocytes.

<p>(a) fresh, (b) freeze-dried lymphocytes as donor cells. Bar = 20 µm.</p