Reproduction Abstracts

The preimplantation embryo undergoes dynamic changes in energy demand during development. Oxygen consumption rate (OCR), representing overall oxidative metabolism, has been reported in several species but few studies have examined the components of OCR. Individual blastocyst OCR was measured using a non-invasive oxygen probe (unisense), whereas OCR of grouped blastocysts was determined using the Oxygen Biosensor fluorometric assay (BD Bioscience). Respiratory chain inhibitors were used to assess different components of OCR. Data was analysed using ANOVA with post-hoc Bonferroni’s test. Addition of 0.2 μM antimycin to inhibit mitochondrial OCR caused mean OCR to fall from 26±4.7 to 2.3±1.3 pmol/embryo per h, indicating that ~87% of OCR was accounted for by mitochondrial activity. Inhibiting ATP synthase with 0.05 μg/ml oligomycin reduced mean OCR from 19.7±2.8 to 6.4±2.3 pmol/embryo per h, meaning that 33% of OCR was uncoupled from ATP synthesis. Addition of the uncoupler 2,4-DNP increased mean OCR from 17.1±2.5 to 32.3±2.7 pmol/embryo per h (P≤0.001). The difference between these values, known as the spare respiratory capacity, was 15.2 pmol/embryo per h. Following inhibition of complex I with 0.01 μM rotenone, mean OCR fell from 27.7±1.6 to 11.6±0.9 pmol/embryo per h, suggesting that 16.1±1.1 pmol/embryo per h was complex I-dependent. Overall, 66% of basal blastocyst OCR was coupled to ATP synthesis, with 58% driven by the NADH-dependent complex I and 8% by the FADH₂-dependent complex II. 21% of basal OCR was uncoupled while non-mitochondrial processes accounted for 13%. A +89% ‘spare’ capacity may enable ATP synthesis to increase in response to high energy demand processes e.g. blastocoel expansion. This profile highlights plasticity in metabolic regulation and allows re-interpretation of existing data.