Antimüllerian hormone inhibits follicle-stimulating hormone-induced adenylyl cyclase activation, aromatase expression, and estradiol production in human.

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Antimüllerian hormone inhibits follicle-stimulating hormone-induced adenylyl cyclase activation, aromatase expression, and estradiol production in human granulosa-lutein cells Hsun-Ming Chang, M.D., Christian Klausen, Ph.D., Peter C.K. Leung, Ph.D., F.R.S.C. Fertility and Sterility Volume 100, Issue 2, Pages 585-592.e1 (August 2013) DOI: 10.1016/j.fertnstert.2013.04.019 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Antimüllerian hormone (AMH) inhibits FSH-stimulated E2 accumulation and aromatase expression in human granulosa-lutein cells. Cells were cultured for 48 hours in absence or presence of AMH (10 ng/mL) while being cotreated with vehicle control or FSH (0.2 IU/mL). Estradiol accumulation was measured by enzyme immunoassay (N = 6) (A) and aromatase messenger RNA (mRNA) (N = 3) (B) and protein (N = 4) (C) levels were measured by reverse transcription quantitative real-time polymerase chain reaction (PCR) and Western blot, respectively. (D) The concentration-dependent effects of AMH (1, 10, 25, or 100 ng/mL) on aromatase mRNA levels were also examined (N = 3). Values (mean ± SEM) without a common letter are significantly different (P<.05). Fertility and Sterility 2013 100, 585-592.e1DOI: (10.1016/j.fertnstert.2013.04.019) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effects of antimüllerian hormone (AMH) on FSH receptor (FSHR) and forskolin- and 8-Br-cyclic adenosine 3′:5′ monophosphate (cAMP)-stimulated aromatase expression. (A, B) Cultures of human granulosa-lutein cells were incubated for 48 hours in the absence or presence of AMH (10 ng/mL) while being cotreated with vehicle control or FSH (0.2 IU/mL), and FSHR messenger RNA (mRNA) (N = 4) (A) and protein (N = 3) (B) levels were measured by reverse transcription quantitative real-time polymerase chain reaction (PCR) and Western blot. (C–F) Cells were cultured for 48 hours in the absence or presence of AMH (10 ng/mL) while being cotreated with vehicle control, 10 μM forskolin (C, D), or 1 mM 8-Br-cAMP (E, F), and changes in aromatase mRNA (N = 5) (C, E) and protein (N = 3) (D, F) levels were measured by reverse transcription quantitative real-time PCR and Western blot. Values (mean ± SEM) without a common letter are significantly different (P<.05). Fertility and Sterility 2013 100, 585-592.e1DOI: (10.1016/j.fertnstert.2013.04.019) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Antimüllerian hormone (AMH) inhibits FSH-stimulated intracellular cyclic adenosine 3′:5′ monophosphate (cAMP) accumulation in human granulosa-lutein cells. Cells were preincubated for 60 minutes with 3-isobutyl-1-methylxanthine (IBMX) and then cultured for 6 hours in the absence or presence of AMH (10 ng/mL) while being cotreated with vehicle control or FSH (0.2 IU/mL), and intracellular cAMP accumulation was measured by enzyme immunoassay (N = 4). Values (mean ± SEM) without a common letter are significantly different (P<.05). Fertility and Sterility 2013 100, 585-592.e1DOI: (10.1016/j.fertnstert.2013.04.019) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Knockdown of type II AMH receptor (AMHR2) abolishes the inhibitory effects of AMH on FSH-induced aromatase expression in hGL cells. (A, B) Cells were transfected for 48 hours with transfection reagent alone (iMAX), control siRNA (siControl), or AMHR2 siRNA (siAMHR2), and AMHR2 messenger RNA (mRNA) (N = 4) (A) and protein (N = 3) (B) levels were measured by reverse transcription quantitative real-time polymerase chain reaction (PCR) and Western blot. (C–E) Cells were treated for 48 hours with siControl or siAMHR2 before being cotreated with vehicle control or FSH (0.2 IU/mL). Changes in aromatase mRNA (N = 3) (C) and protein (N = 4) (E) levels were measured by reverse transcription quantitative real-time PCR and Western blot. (D) Knockdown effects of AMHR2 after transfection of siControl or siAMHR2. AMHR2 mRNA levels were measured by reverse transcription quantitative real-time PCR. Values (mean ± SEM) without a common letter are significantly different (P<.05). si = small interfering. Fertility and Sterility 2013 100, 585-592.e1DOI: (10.1016/j.fertnstert.2013.04.019) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions