Human granulosa luteal cell oxidative phosphorylation function is not affected by age or ovarian response

Coenzyme Q–dependent mitochondrial respiratory chain activity in granulosa cells is reduced with aging

“…Moreover, we demonstrate that the decrease in mitochondrial function is not general but involves CoQ 10 -dependent enzymes. This stands along with our previous study that found no change in cytochrome c oxidase activity (complex IV, not dependent on CoQ 10 ) with aging (15). Because MRC electron transport is a chain reaction, a decrease in one step can become a rate-limiting step.…”

“…This could also indicate decreased respiratory chain activity per mitochondria in older women while the total activity remains constant in the cell. However, our previous study (15) did not detect any difference in cytochrome c oxidase (respiratory chain complex IV) activity relative to CS. Thus, this issue remains open for in-depth future studies.…”

“…The enzymatic activity was measured in the homogenates after a 5-second sonication at 37 C by spectrophotometry as we have described elsewhere (15). Complex II was measured as succinate dehydrogenase (SDH) based on the succinatemediated phenazine methosulfate reduction of dichloroindophenol at 600 nm.…”

“…Although white blood cell contamination might be of concern, it was previously calculated and reported (15) that over 90% of the homogenate protein originates from the GC. Notably we did not isolate mitochondria because considerable material is lost during isolation and there is frequently insufficient GC from a single patient (especially in poor responders) to obtain enough mitochondria for analysis.…”

Cap-independent translation by DAP5 controls cell fate decisions in human embryonic stem cells

“…The homogenate was centrifuged at 2000g for 3 min at 4°C to remove nuclei, and the resulting supernatant was centrifuged at 12,000g for 10 min at 4°C to pellet mitochondria, which were stored in isolation buffer at −70°C until use. The enzymatic activity of respiratory chain complex I (rotenone-sensitive NADH-CoQ reductase) was measured as reported previously (Shufaro et al 2012) by monitoring the oxidation of NADH by spectrophotometry at 340 nm at 37°C in the presence of coenzyme Q 1 . Activity of the mitochondrial matrix enzyme citrate synthase, used as a control, was measured in the presence of acetyl-CoA and oxaloacetate by monitoring the release of CoASH coupled to 5 ′ ,5 ′ -dithiobis (2-nitrobenzoic) acid at 412 nm.…”