The human cumulus cell transcriptome provides poor predictive value for embryo transfer outcome.

RESEARCH QUESTION: Is the transcriptome of cumulus cells a good predictor of the embryo's developmental competence? DESIGN: Cumulus cells were collected from donor oocytes and their transcriptome was analysed by RNA sequencing analysis at >30 × 106 reads in samples grouped according to the developmental potential of their enclosed oocyte: not able to develop to the blastocyst stage (Bl-), able to develop to the blastocyst stage but failing to establish a pregnancy (P-), or able to develop to the blastocyst stage and to establish a clinical pregnancy (P+). RESULTS: The cumulus cell trancriptome was largely independent of the developmental potential as, using a false dscovery rate-adjusted P-value of <0.05, only 10, 11 and 5 genes were differentially expressed for the comparisons P+ versus P-, P+ versus Bl-, and P- versus Bl-, respectively, out of a total of 17,469 genes expressed. Between the differentially expressed genes, those showing little overlap between samples from different groups were CHAC1, up-regulated in the P- and P+ groups compared with the Bl- group, and CENPE, CD93, PECAM1 and HSPA1B, which showed the opposite expression pattern. Focusing on the pregnancy potential, only EPN3 was consistently downregulated in the P+ compared with the P- and Bl- groups. CONCLUSIONS: The cumulus cell transcriptome is largely unrelated to the establishment of clinical pregnancy following embryo transfer, although the expression level of a subset of genes in cumulus cells may indicate the ability to develop to the blastocyst stage.

Kinetic model of the inhibition of respiration by endogenous nitric oxide in intact cells.

Nitric oxide (NO) inhibits mitochondrial respiration by decreasing the apparent affinity of cytochrome c oxidase (CcO) for oxygen. Using iNOS-transfected HEK 293 cells to achieve regulated intracellular NO production, we determined NO and O(2) concentrations and mitochondrial O(2) consumption by high-resolution respirometry over a range of O(2) concentrations down to nanomolar. Inhibition of respiration by NO was reversible, and complete NO removal recovered cell respiration above its routine reference values. Respiration was observed even at high NO concentrations, and the dependence of IC(50) on [O(2)] exhibits a characteristic but puzzling parabolic shape; both these features imply that CcO is protected from complete inactivation by NO and are likely to be physiologically relevant. We present a kinetic model of CcO inhibition by NO that efficiently predicts experimentally determined respiration at physiological O(2) and NO concentrations and under hypoxia, and accurately predicts the respiratory responses under hyperoxia. The model invokes competitive and uncompetitive inhibition by binding of NO to the reduced and oxidized forms of CcO, respectively, and suggests that dissociation of NO from reduced CcO may involve its O(2)-dependent oxidation. It also explains the non-linear dependence of IC(50) on O(2) concentration, and the hyperbolic increase of c(50) as a function of NO concentration.

Relative sensitivity of soluble guanylate cyclase and mitochondrial respiration to endogenous nitric oxide at physiological oxygen concentration.

Nitric oxide (NO) is a widespread biological messenger that has many physiological and pathophysiological roles. Most of the physiological actions of NO are mediated through the activation of sGC (soluble guanylate cyclase) and the subsequent production of cGMP. NO also binds to the binuclear centre of COX (cytochrome c oxidase) and inhibits mitochondrial respiration in competition with oxygen and in a reversible manner. Although sGC is more sensitive to endogenous NO than COX at atmospheric oxygen tension, the more relevant question is which enzyme is more sensitive at physiological oxygen concentration. Using a system in which NO is generated inside the cells in a finely controlled manner, we determined cGMP accumulation by immunoassay and mitochondrial oxygen consumption by high-resolution respirometry at 30 microM oxygen. In the present paper, we report that the NO EC50 of sGC was approx. 2.9 nM, whereas that required to achieve IC50 of respiration was 141 nM (the basal oxygen consumption in the absence of NO was 14+/-0.8 pmol of O2/s per 10(6) cells). In accordance with this, the NO-cGMP signalling transduction pathway was activated at lower NO concentrations than the AMPKs (AMP-activated protein kinase) pathway. We conclude that sGC is approx. 50-fold more sensitive than cellular respiration to endogenous NO under our experimental conditions. The implications of these results for cell physiology are discussed.