Embryonic diapause is conserved across mammals

Abstract

Embryonic diapause (ED) is a temporary arrest of embryo development and is characterized by delayed implantation in the uterus. ED occurs in blastocysts of less than 2% of mammalian species, including the mouse (Mus musculus). If ED were an evolutionarily conserved phenomenon, then it should be inducible in blastocysts of normally non-diapausing mammals, such as domestic species. To prove this hypothesis, we examined whether blastocysts from domestic sheep (Ovis aries) could enter into diapause following their transfer into mouse uteri in which diapause conditions were induced. Sheep blastocysts entered into diapause, as demonstrated by growth arrest, viability maintenance and their ED-specific pattern of gene expression. Seven days after transfer, diapausing ovine blastocysts were able to resume growth in vitro and, after transfer to surrogate ewe recipients, to develop into normal lambs. The finding that non-diapausing ovine embryos can enter into diapause implies that this phenomenon is phylogenetically conserved and not secondarily acquired by embryos of diapausing species. Our study questions the current model of independent evolution of ED in different mammalian orders.

Figure 1. Experimental design of embryonic diapause (ED) induction in ovine blastocysts by transfer into ovariectomised pseudo-pregnant mice at 2.5 dpc.

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.

Figure 2. Confirmation of ED in ovine embryos.

(a) Proportion of BrdU- and TUNEL-positive cells in diapausing ovine and murine blastocysts flushed from ovariectomised mouse uteri and in controls (b) qRT-PCR analysis of genes involved in ED control. Genes that positively regulate cell proliferation (PCNA) and signaling (HB-EGF) were not expressed in diapausing ovine blastocysts, while the anti-proliferative gene BTG1 was significantly over-expressed. IGF2R 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. Results are mean ± S.E.M. *** p<0.0001, ** p<0.003, *p<0.03.

Figure 3. Reversibility of growth arrest in ovine embryos following flushing from the uterus of ovariectomised mice

. (a) Ovine blastocysts before (i) and immediately after (ii) transfer to mouse uteri, and following 12 hours of culture in vitro (iii). (b) Percentage of BrdU-positive, proliferating cells and of embryos hatching from the zona pellucida 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. Results are mean ± S.E. M *p<0.05.