Early developmental indices of placental growth restriction in the overfed adolescent sheep
RG Lea1,3, LT Hannah1, D Redmer2, PA Fowler3, J Murray4, R Aitken1, JS Milne1 & JM Wallace1
Introduction: Overfeeding adolescent ewes with singleton pregnancies results in reduced placental size and low lamb birth weight at term. We have investigated if reduced placental size is associated with altered placental cell proliferation and apoptosis at day 80 of pregnancy (peak placental growth period). Methods: Singleton pregnancies to a single sire were established by embryo transfer to adolescent recipient dams. Pregnant ewes were offered high (H, n = 14) or moderate (M, n = 13) diets predicted to induce restricted and normal placental size at term. Animals (n=8/group) were administered Brd-U 1h prior to slaughter (day 80). NBF fixed or frozen placental samples were subjected to immunohistochemistry or in-situ hybridisation for markers of (a) proliferation: Brd-U, PCNA (b) apoptosis regulatory genes: bcl-2 and bax protein, mcl-1 and bax mRNA. Results were quantified by image analysis. Results: Total placentome weights were not significantly different (H:703 ± 48 v M:658 ± 36). Brd-U and PCNA were detected predominantly in the trophectoderm. Brd-U immunostaining was significantly lower in the high intake group (H:9.0 ± 0.9 v M:12.6 ± 0.7, P<0. 01). PCNA was significantly increased in the high intake group likely indicative of placental damage (H:4.68 ± 0.50 v M:2.31 ± 0.50: P<0.05). Bax mRNA and protein as well as mcl-1 mRNA were predominantly localised to the trophectoderm. Bax protein was specifically localised to the maternal aspect of the uninucleate trophectoderm cells and by Western blot, was significantly increased in the high intake group (band volume H:50,804 ± 2,399 v M:21,040 ± 4046: P<0.001). Low level bcl-2 was localised to uninucleate and binucleate cells. Conclusion: Placental growth in day 80 overfed adolescent ewes is associated with reduced trophectoderm proliferation and increased bax. These changes occur before differences in placental size are apparent and may be early indicators of nutritionally- mediated placental growth restriction.