Expression and localization of opioid receptors during the maturation of human oocytes Ekaitz Agirregoitia, Laura Peralta, Rosario Mendoza, Antonia Expósito,

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Expression and localization of opioid receptors during the maturation of human oocytes Ekaitz Agirregoitia, Laura Peralta, Rosario Mendoza, Antonia Expósito, Elena Díaz Ereño, Roberto Matorras, Naiara Agirregoitia Reproductive BioMedicine Online Volume 24, Issue 5, Pages 550-557 (May 2012) DOI: 10.1016/j.rbmo.2012.02.007 Copyright © 2012 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Reverse-transcription PCR products for δ- (OPRD1), κ- (OPRK1) and μ- (OPRM1) opioid receptors and β-actin (ACTB) in each maturation stage of human oocytes (germinal vesicle (GV), metaphase I (MI) and metaphase II (MII)) and grey matter from the human prefrontal cerebral cortex (Ctx). Products were separated by electrophoresis on 2% agarose gel and stained with ethidium bromide. Experiments were performed in triplicate; representative results are shown. Reproductive BioMedicine Online 2012 24, 550-557DOI: (10.1016/j.rbmo.2012.02.007) Copyright © 2012 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Changes in expression of δ- (OPRD1), κ- (OPRK1) and μ- (OPRM1) opioid receptor genes relative to ACTB in the human oocyte (Oct) and the grey matter from the human prefrontal cerebral cortex (Ctx) using specific primers. The relative concentrations of opioid receptor mRNA were determined by quantitative reverse-transcription PCR. Cycle threshold values were normalized to ACTB and the average ΔCt of the cortex sample was used for calibration. Each value is the mean±SEM of three replicates. Reproductive BioMedicine Online 2012 24, 550-557DOI: (10.1016/j.rbmo.2012.02.007) Copyright © 2012 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Western blotting analysis of δ- (OPRD1), κ- (OPRK1) and μ- (OPRM1) opioid receptors in human oocytes (Oct) and grey matter from the human prefrontal cerebral cortex (Ctx) using specific rabbit antiserum. Oocytes from all maturational stages were pooled in order to provide enough material for immunoblotting. Molecular weights (MW; kDa) are indicated on the left. Experiments were performed in triplicate; representatives of Western blots obtained with ∼60 oocytes are shown. Reproductive BioMedicine Online 2012 24, 550-557DOI: (10.1016/j.rbmo.2012.02.007) Copyright © 2012 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Immunofluorescence analysis of δ- (OPRD1), κ- (OPRK1) and μ- (OPRM1) opioid receptors in each maturation stage of human oocytes (germinal vesicle (GV), metaphase I (MI) and metaphase II (MII)). The equatorial plane for each oocyte, which was scanned from top to bottom, is represented. Experiments were performed with 15 replicates per stage; representative results are shown. Bar=40μm. Reproductive BioMedicine Online 2012 24, 550-557DOI: (10.1016/j.rbmo.2012.02.007) Copyright © 2012 Reproductive Healthcare Ltd. Terms and Conditions