Dynamics of expression and localization of the cannabinoid system in granulosa cells during oocyte nuclear maturation Ekaitz Agirregoitia, Ph.D., Inés.

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Dynamics of expression and localization of the cannabinoid system in granulosa cells during oocyte nuclear maturation Ekaitz Agirregoitia, Ph.D., Inés Ibarra-Lecue, B.S., Lide Totorikaguena, B.S., Rosario Mendoza, B.S., Antonia Expósito, Ph.D., Roberto Matorras, M.D., Ph.D., Leyre Urigüen, Ph.D., Naiara Agirregoitia, Ph.D. Fertility and Sterility Volume 104, Issue 3, Pages 753-760 (September 2015) DOI: 10.1016/j.fertnstert.2015.06.013 Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 1 The mRNA expression of (A) CB1, (B) CB2, (C) FAAH, and (D) MGLL in human granulosa cells (Gra) from GV, MI, and MII oocytes using specific primers. The relative concentrations of protein mRNA were determined by quantitative RT-PCR. Cycle threshold values were normalized to GAPDH, and the average ΔCt of the cortex sample (for CB1, FAAH, and MGLL) and Jurkat cells (for CB2) were used for calibration. Each value is the mean (±SEM) of three replicates. Fertility and Sterility 2015 104, 753-760DOI: (10.1016/j.fertnstert.2015.06.013) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Immunofluorescence analysis of CB1, CB2, FAAH, and MGLL in human granulosa cells from GV, MI, and MII oocytes. The distribution of cannabinoid receptors and cannabinoid-degrading enzymes is shown in green. A negative control consisting of preadsorption of primary antibody with each specific blocking peptide is shown. Hoechst-labeled DNA is shown in blue. n = 15 per stage. Representative photomicrographs are shown. The scale bar represents 10 μm. Fertility and Sterility 2015 104, 753-760DOI: (10.1016/j.fertnstert.2015.06.013) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Western blotting analysis of CB1 and CB2 receptor, FAAH, and MGLL in human granulosa cells (Gra), gray matter from the human prefrontal cerebral cortex (Cx), and Jurkat cells (Jkt) using a rabbit antiserum against CB1, CB2, FAAH, and MGLL. Molecular weights (MW-kDa) are indicated on the left; n = 3; representative Western blots are shown. Fertility and Sterility 2015 104, 753-760DOI: (10.1016/j.fertnstert.2015.06.013) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Immunofluorescence analysis of (A) CB2, (B) FAAH, and (C) MGLL compared with CB1 cannabinoid receptor in human granulosa cells. The distributions of CB2, FAAH, and MGLL are shown in green, and the distribution of CB1 is shown in red. Their overlap is shown in yellow. Hoechst-labeled DNA is shown in blue. n = 15 per stage. Representative photomicrographs are shown. The scale bar represents 10 μm. Fertility and Sterility 2015 104, 753-760DOI: (10.1016/j.fertnstert.2015.06.013) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions