Resistin decreases insulin-like growth factor I–induced steroid production and insulin- like growth factor I receptor signaling in human granulosa cells  Maxime Reverchon, M.S., Marion Cornuau, M.D., Christelle Ramé, B.Sc., Fabrice Guerif, Ph.D., Dominique Royère, M.D., Joëlle Dupont, Ph.D.  Fertility and Sterility  Volume 100, Issue 1, Pages 247-255.e3 (July 2013) DOI: 10.1016/j.fertnstert.2013.03.008 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Measurement of the concentration of resistin in plasma and follicular fluid of women treated with IVF. Plasma (♦) and FF (○) resistin levels after collecting blood and follicular fluid on the day of the oocyte retrieval from nine infertile women undergoing IVF. Each concentration was determined by ELISA as described in the Materials and Methods section. The individual data (A) or the mean of these data (B) are represented. *P<.05. Fertility and Sterility 2013 100, 247-255.e3DOI: (10.1016/j.fertnstert.2013.03.008) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Expression of resistin in human ovarian follicle. (A) Total RNA from fresh human GCs, cumulus cells obtained from women undergoing IVF, and a human ovarian granulosa tumor-derived cell line (KGN) was extracted, as described in Materials and Methods. We performed RT-PCR with primers designed to amplify fragments of resistin (300 pb). Human visceral (AT Vis) and subcutaneous (AT Sc) adipose tissues were used as a positive control for resistin expression (mRNA [A] and protein [B]). Tissues or cells from four different patients were used and two replications per patient were performed. (B) Protein extracts (50 μg) were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with specific antibodies against resistin. Equal protein loading was verified by reprobing membranes with an antivinculin antibody. Tissues or cells from four different patients were used, and two replications per patient were performed. (C) Localization of resistin in human ovarian follicles by immunohistochemistry. The two panels at the bottom are higher magnifications of part of the panels on the top. We performed DAB-immunoperoxidase staining on paraffin-embedded human ovary using antibodies against resistin (2, 4, 5, and 6), or no primary antibodies but rabbit IgG (1 and 3). The immunospecific staining is brown. The sections were counterstained with hematoxylin. Resistin was detected in the granulosa cells (GCs) and theca cells (T) of large follicles and in GCs and oocyte (Oo) in primary follicle. A: antrum. Bars = 100 μm or 20 μm. Immunohistochemical analysis was performed on two different human ovary slides from each of four patients. Fertility and Sterility 2013 100, 247-255.e3DOI: (10.1016/j.fertnstert.2013.03.008) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Effect of recombinant human resistin on basal and FSH or insulin-like growth factor I (IGF-I)-stimulated P (A) and E2 (B) secretions by primary human GCs and on the amount of the P450scc (C) and P450 aromatase (D) protein in primary human GCs. Human GCs were cultured for 48 hours in a medium with serum and then in serum-free medium in the absence or presence of resistin (10 g/mL) ± FSH (10−8 M) or IGF-I (10−8 M) as described in the Materials and Methods section. The culture medium was collected and P (A) and E2 (B) production was measured by radioimmunoassay. Results are mean ± standard error of the mean (SEM) of the three independent groups of GCs from five or six patients. Different letters indicate statistically significant differences (P<.05). (C, D) Protein extracts from human GCs, cultured for 48 hours in the absence or presence of resistin (10 ng/mL) ± FSH (10−8 M) or insulin-like growth factor I (IGF-I) (10−8 M), were submitted to SDS-PAGE, as described in the Materials and Methods section. The membranes were probed with antibodies against P450scc (C) and P450 aromatase (D). Equal protein loading was verified by reprobing membranes with an antivinculin antibody. Results are representative of at least three independent experiments. The blots were quantified, and the P450scc or P450 aromatase-to-vinculin ratio is shown. The results are expressed as mean ± standard error of the mean (SEM). Different letters indicate statistically significant differences (P<.05). Fertility and Sterility 2013 100, 247-255.e3DOI: (10.1016/j.fertnstert.2013.03.008) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effect of human recombinant resistin on the MAPK ERK1/2 (A), p38 (B), and Akt (C) phosphorylation levels and on phosphorylation of IGF-IR β subunit (D) and MAPK ERK1/2 (E) in response to IGF-I in primary human GCs. Human GC lysates were prepared from cells incubated with 10 ng/mL resistin for various times: 0, 5, 10, 30, 60, or 120 minutes. Lysates (50 mg) were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-phospho-MAPK ERK1/2 (A), p38 (B), or Akt (C) and then with anti-ERK2, p38 or Akt total protein antibodies. Representative blots from three independent experiments are shown. Blots were quantified, and the phosphorylated MAPK ERK1/2/ERK2, phosphorylated MAPK p38/p38 or phosphorylated Akt protein ratios are shown. The results are represented as mean ± standard error of the mean (SEM). Different letters indicate statistically significant differences (P<.05). (D, E) Primary human GCs were cultured in a medium with serum and then in serum-free medium in the absence or in the presence of resistin (10 ng/mL) ± IGF-I (10−8 M) for 48 hours (conditions used to measure P and E2 production). Cells were lysed, and lysates were directly subjected to immunoblotting with antibodies recognizing phosphotyrosine (PY20) (panel D) or with anti-phospho-MAPK ERK1/2 (panel E) antibodies. IGF-IRβ and MAPK ERK2 levels were evaluated by reprobing the membranes with IGF-IRβ and ERK2 total antibodies, respectively. Representative blots from four different cultures are shown. Each culture was performed by using cells obtained from different follicles from one patient. In each culture, each treatment (resistin in the presence or absence of IGF-I) was applied in duplicate. The blots were quantified, and the phosphorylated protein/total protein ratio is shown. The results are represented as mean ± SEM. Fertility and Sterility 2013 100, 247-255.e3DOI: (10.1016/j.fertnstert.2013.03.008) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Effect of recombinant human resistin in the presence or absence of IGF-IRβ 1H7 antibody on basal and FSH or insulin-like growth factor I (IGF-I)-stimulated P (A) and E2 (B) secretions by primary human GCs. Human GCs were cultured for 48 hours in a medium with serum and then in serum-free medium in the absence or presence of resistin (10 g/mL) ± IGF-I (10−8 M) ± IGF-IRβ IH7 antibody (10 μg/mL), as described in the Materials and Methods section. The culture medium was collected, and P (A) and E2 (B) production was measured by radioimmunoassay. Results are mean ± standard error of the mean (SEM) of the three independent groups of GCs from five or six patients. Different letters indicate statistically significant differences (P<.05). Fertility and Sterility 2013 100, 247-255.e3DOI: (10.1016/j.fertnstert.2013.03.008) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Effect of human recombinant resistin in the presence or in the absence of IGF-IRβ IH7 antibody on phosphorylation of IGF-IR β subunit (A) and MAPK ERK1/2 (B) in response to IGF-I in primary human GCs. Primary human GCs were cultured in a medium with serum and then in serum-free medium in the absence or in the presence of resistin (10 ng/mL) ± IGF-I (10−8 M) ± IGF-IR 1H7 antibody (10 μg/mL) for 48 hours (conditions used to measure P and E2 production). Cells were lysed, and the lysates were directly subjected to immunoblotting with antibodies recognizing phosphotyrosine (PY20) (panel A) or with anti-phospho-MAPK ERK1/2 (panel B) antibodies. The IGF-IRβ and MAPK ERK2 levels were evaluated by reprobing the membranes with IGF-IRβ and ERK2 total antibodies, respectively. Representative blots from four different cultures are shown. Each culture was performed by using cells obtained from different follicles from one patient. In each culture, each treatment (resistin in the presence or absence of IGF-I ± IGF-IR 1H7 antibody) was applied in duplicate. The blots were quantified, and the phosphorylated protein/total protein ratio is shown. The results are represented as mean ± SEM. Fertility and Sterility 2013 100, 247-255.e3DOI: (10.1016/j.fertnstert.2013.03.008) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions