Rong Hu, Ph. D. , Fei-miao Wang, M. D. , Liang Yu, Ph. D. , Yan Luo, M

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Antimüllerian hormone regulates stem cell factor expression in human granulosa cells Rong Hu, Ph.D., Fei-miao Wang, M.D., Liang Yu, Ph.D., Yan Luo, M.D., Xin Wu, B.Sc., Juan Li, M.D., Xiao-mei Zhang, Ph.D., Sergio Oehninger, M.D., Ph.D., Silvina Bocca, M.D., Ph.D. Fertility and Sterility Volume 102, Issue 6, Pages 1742-1750.e1 (December 2014) DOI: 10.1016/j.fertnstert.2014.08.012 Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A) Correlation between protein expression levels of antimüllerian hormone (AMH) and stem cell factor (SCF) in follicular fluid (FF) from small follicles. ELISA was used to analyze AMH and SCF concentration in FF from IVF patients (n = 50). Results showed that there was a statistically significant negative correlation (r = −0.65; P<.05). (B) Correlation between the mRNA expression levels of AMH and SCF in GCs from small follicles of IVF patients (n = 31). Real-time PCR was used to analyze AMH and SCF mRNA in human GCs. Results showed there was a statistically significant negative correlation (r = −0.79; P<.01). Fertility and Sterility 2014 102, 1742-1750.e1DOI: (10.1016/j.fertnstert.2014.08.012) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Stem cell factor (SCF) mRNA and protein expression were regulated by recombinant human antimüllerian hormone (AMH) in human granulosa cells (GCs) in a dose-dependent fashion. (A) Real-time PCR was used to analyze mRNA expression of SCF. Each bar indicates the fold decrease of mRNA expression relative to the housekeeping gene 18S. Results showed SCF mRNA expression was down-regulated by recombinant human AMH (overall effect P<.01). At 15 ng/mL and 20 ng/mL, the results were statistically significantly lower than control (P<.05 and P<.001, respectively). (B) SCF protein expression in the cell lysates after 48 hours of treatment with recombinant human AMH were demonstrated by immunoblotting. Stem cell factor protein expression was down-regulated by recombinant human AMH (overall effect P<.01); at 15 ng/mL and 20 ng/mL, the recombinant human AMH results were statistically significantly lower than control (P<.05 and P<.01, respectively). Fertility and Sterility 2014 102, 1742-1750.e1DOI: (10.1016/j.fertnstert.2014.08.012) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 3 (A) Stem cell factor (SCF) mRNA regulation by cyclic adenosine 3′:5′ monophosphate (cAMP) in human granulosa cells (GCs). After seeding, cells were cultured for 48 hours in control medium or in the presence of different cAMP concentrations (1–4 nM). Real-time PCR results showed that cAMP statistically significantly stimulated SCF mRNA expression in a dose-dependent fashion (overall effect P<.01). At 3 and 4 nM, cAMP strongly stimulated SCF mRNA expression (both P<.001 vs. control). (B) Immunolocalization of SCF in human GCs cultured for 48 hours with control medium (i), cAMP (ii), recombinant human antimüllerian hormone (AMH) (iii), and combination of recombinant human AMH and cAMP (iv) (magnification: ×400). NC = negative control (nonimmune mouse IgG). Stem cell factor was expressed in the cytoplasm of human GCs. Stem cell factor had limited and lower expression in the recombinant human AMH group (P<.05 vs. controls). In contrast, a very strong and statistically significantly higher staining was observed in the human GCs treated with cAMP (P<.01 vs. control). There was no difference in SCF expression in the recombinant human AMH and cAMP group compared with control conditions. Fertility and Sterility 2014 102, 1742-1750.e1DOI: (10.1016/j.fertnstert.2014.08.012) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 4 (A) Human granulosa cells (GCs) were cultured for 48 hours in control medium or in the presence of antimüllerian hormone (AMH) (20 ng/mL), cyclic adenosine 3′:5′ monophosphate (cAMP) (3 nM), recombinant human AMH and cAMP, recombinant human AMH and H89 (1 mM), or cyclic adenosine 3′:5′ monophosphate (cAMP) and H89 (1 mM). The results of real-time PCR (gene expression) and immunoblotting (protein expression) were consistent (A and B, respectively). As expected, recombinant human AMH significantly reduced mRNA and protein expression of stem cell factor (SCF) (both P<.01 vs. control), and cAMP statistically significantly increased mRNA and protein expression of SCF (P<.001 and P<.01, respectively, vs. control). Furthermore, recombinant human AMH statistically significantly reduced the effect of cAMP on SCF mRNA and protein expression (cAMP vs. recombinant human AMH and cAMP, P<.001 and P<.01). Using an inhibitor of protein kinase A (PKA) (H89), we then tested whether activation of the cAMP/PKA pathway regulating SCF was suppressed by recombinant human AMH. H89 significantly inhibited the effects of both recombinant human AMH and cAMP on SCF gene and protein expression (recombinant human AMH vs. recombinant human AMH and H89, both P<.05; cAMP vs. cAMP and H89, P<.01 and P<.001, respectively). Fertility and Sterility 2014 102, 1742-1750.e1DOI: (10.1016/j.fertnstert.2014.08.012) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Study flow chart for first experiments. A total of 197 follicular fluid (FF) samples from 78 patients who underwent IVF-ET were collected for the first experiments. Among them, 83 FF samples from 50 patients were not grossly contaminated with blood and were used for ELISA. We discarded 114 FF samples because of blood contamination (37 FF samples and 77 samples from 50 patients and 28 patients, respectively). Thirty-one GCs samples were collected for RT-PCR to study the relationship between the antimüllerian hormone (AMH) and stem cell factor (SCF) mRNA. Fertility and Sterility 2014 102, 1742-1750.e1DOI: (10.1016/j.fertnstert.2014.08.012) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions