Expression and possible implication of growth hormone–releasing hormone receptor splice variant 1 in endometriosis Li Fu, Ph.D., M.D., Yutaka Osuga,

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Expression and possible implication of growth hormone–releasing hormone receptor splice variant 1 in endometriosis Li Fu, Ph.D., M.D., Yutaka Osuga, Ph.D., M.D., Tetsu Yano, Ph.D., M.D., Yuri Takemura, Ph.D., M.D., Chieko Morimoto, Ph.D., M.D., Yasushi Hirota, Ph.D., M.D., Andrew V. Schally, Ph.D., M.D.h.c., Yuji Taketani, Ph.D., M.D. Fertility and Sterility Volume 92, Issue 1, Pages 47-53 (July 2009) DOI: 10.1016/j.fertnstert.2008.04.048 Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Reverse transcriptase polymerase chain reaction analysis of the splice variants of the growth hormone–releasing hormone (GHRH) receptor and GHRH gene expression in eutopic and ectopic endometrial tissues. Data represent eutopic and ectopic endometrial tissues from 27 women with endometriosis, and eutopic endometrial tissues from 20 women without endometriosis. Amplification of GAPDH was performed to ensure equal loading. Lane M: 100-bp DNA molecular marker. Lanes 1–3: eutopic endometrial tissues from different patients without endometriosis. Lanes 4–6: eutopic endometrial tissues from different patients with endometriosis. Lanes 7–10: ectopic endometrial tissues from different patients. Fertility and Sterility 2009 92, 47-53DOI: (10.1016/j.fertnstert.2008.04.048) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effect of exogenous hGHRH(1-29)NH2 on cAMP production in cultured ectopic endometrial stromal cells (ESC). The cells were treated for 2 hours with hGHRH(1-29)NH2 (10−9 to 10−6 M) or forskolin (10−7 M). The cells were co-treated with isobutylmethylxanthine (IBMX) to suppress cAMP degradation. Conditioned media were assayed for cAMP concentration, and the cell numbers were determined using the Cell Counting Kit-8. Data represent the mean ± standard error of the mean of cAMP concentration per cell number of four separate experiments using different ESC preparations, expressed as a percentage of untreated controls. ∗P<.05 versus control. Fertility and Sterility 2009 92, 47-53DOI: (10.1016/j.fertnstert.2008.04.048) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Effect of hGHRH(1-29)NH2 on the proliferation of ectopic endometrial stromal cells (ESC) determined by BrdU incorporation using a cell proliferation ELISA system. The ESC were treated with hGHRH(1-29)NH2 (10−7 M and 10−6 M) for 24 hours and 48 hours. Data represent the mean percentage of the untreated control ± standard error of the mean of five independent experiments using hexaplicate wells. ∗P<.05. ∗∗P<.01 versus control. Fertility and Sterility 2009 92, 47-53DOI: (10.1016/j.fertnstert.2008.04.048) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions