298. Dietary protein does not influence mitochondrial distribution in the 2-cell mouse embryo

Abstract

Excess dietary protein can negatively influence fertility. The underlying mechanisms remain to be completely elucidated; however, variations in reproductive tract pH, and ammonium and urea concentrations have been implicated. Mouse embryos cultured in the presence of ammonium showed a shift in mitochondrial distribution away from the nucleus towards the cell cortex, suggestive of reduced mitochondrial activity, ATP production and embryo viability. In this study we determined the effect of dietary protein in vivo, on mitochondrial distribution in the 2-cell mouse embryo. Five-week-old Swiss female mice (n = 10) were fed low (9%), medium (14%) or high (25%) dietary protein for 3 weeks; feed intake and body weight were recorded weekly. At 8 weeks of age mice were primed with 5 IU of PMSG, then 5 IU hCG 48 h later, and mated overnight with males of proven fertility. Forty hours post-hCG females were sacrificed, their oviducts collected and flushed with media. The total number of 2-cell embryos and oocytes retrieved were recorded. Active mitochondria were stained in the 2-cell embryos using Mitotracker Green (Molecular Probes), and were visualised using confocal microscopy. Density of perinuclear and cortical staining was determined in Photoshop 7.0, using an established method. Females fed the medium diet consumed significantly less and gained less weight than those fed the low or high diet (Table 1), despite similar final body weights (data not shown). Females fed the low diet tended to have a lower ovulation rate and fewer 2-cell embryos than females consuming the other diets (Table 1, P > 0.05). There was no significant effect of dietary protein on the distribution of mitochondria between the perinuclear and cortical region of the embryo, which may be reflective of lower in vivo ammonium levels compared to those described in culture.