1. Adiponectin increases insulin-like growth factor I-induced progesterone and estradiol secretion in human granulosa cells 
Christine Chabrolle, M.D., Lucie Tosca, Ph.D., Christelle Ramé, Technician, Pierre Lecomte, M.D., Dominique Royère, M.D., Joëlle Dupont, Ph.D. 
Fertility and Sterility 
Volume 92, Issue 6, Pages (December 2009)
DOI: /j.fertnstert
Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Measurement of the concentration of adiponectin in plasma and follicular fluid (FF) of women treated with IVF. Serum (□) and FF (■) adiponectin levels after collecting blood and FF of six infertile women undergoing IVF on the day of oocyte retrieval. Each concentration was determined by ELISA method 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  , DOI: ( /j.fertnstert )
Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
Messenger RNA (A) and protein (B) expression of adiponectin and its receptors AdipoR1 and AdipoR2 in human granulosa (GC) and theca cells. (A) Total RNA from human GC (HG) and theca (HT) cells was extracted as described in the Materials and Methods section and reverse transcriptase–polymerase chain reaction (RT-PCR) was performed by using primers designed to amplify fragments of adiponectin (221 pb), AdipoR1 (500 pb), and AdipoR2 (440 pb). Human ovarian granulosa tumorderived cell line (KGN), human visceral (ATv) and subcutaneous (ATsc) adipose tissue were used as a negative and positive control for adiponectin. (B) Protein extracts (50 μg) were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane, and incubated with specific antibodies against adiponectin, AdipoR1, or AdipoR2. Equal protein loading was verified by reprobing membrane with an antivinculin antibody. Human ATv, human muscle (Mus), liver (Liv), and KGN lysates were used as a positive and negative control. Results are representative of at least three independent experiments.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
Effect of recombinant human adiponectin on basal and FSH or insulin-like growth factor I (IGF-1)-stimulated P (A) and E2 (B) secretions by human granulosa cells (GC). Human GCs were cultured for 48 hours in medium with serum and then in serum-free medium in the absence or in the presence of adiponectin (5 μg/mL) ± FSH (10-8 M) or IGF-1 (10-8 M) as described in the Materials and Methods section. The culture medium was collected and P (A) and E2 (B) productions were measured by RIA. Results are means ± standard error of the mean (SEM) of the three independent groups of GCs from five or six patients. Different letters indicate significant differences (P<.05).
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

5. Figure 4
Effect of human recombinant adiponectin on the amount of the StAR (A) and p450 aromatase (B) protein in human granulosa cells (GC). Protein extracts from human GCs, cultured for 48 hours in the absence or in the presence of adiponectin (5 μg/mL) ± FSH (10-8 M) or insulin-like growth factor I (IGF-1) (10-8 M), were submitted to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) as described in the Materials and Methods section. The membranes were probed with antibodies against StAR (A) and p450 aromatase (B). Equal protein loading was verified by reprobing membranes with an antivinculin antibody. Results are representative of at least three independent experiments. Blots were quantified and the StAR or p450 aromatase-to-vinculin ratio was shown. The results are expressed as mean ± standard error of the mean (SEM). Different letters indicate significant differences (P<.05).
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

6. Figure 5
Effect of human recombinant adiponectin on human granulosa cells (GC) proliferation. Thymidine incorporation was determined in human GCs cultured for 24 hours in the presence or absence of adiponectin (5 μg/mL) ± FSH (10-8 M) or insulin-like growth factor I (IGF-1) (10-8 M) as described in the Materials and Methods section. Results are representative of at least three independent experiments. The results are expressed as means ± standard error of the mean (SEM). Different letters indicate significant differences (P<.05).
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

7. Figure 6
Effect of human recombinant adiponectin on the MAPK ERK1/2 (A) and p38 (B) phosphorylation levels in human granulosa cells (GC). Human GC lysates were prepared from cells incubated with 5 μg/mL adiponectin for various times: 0, 5, 10, 30, 60, or 120 minutes. Lysates (50 μg) were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membrane, and probed with anti-phospho-MAPK ERK1/2 (A) or p38 (B) and then with anti-ERK2 or p38 protein antibodies. Representative blots from three independent experiments are shown. Blots were quantified and the phosphorylated MAPK ERK1/2/ERK2 or the phosphorylated MAPK p38/p38 protein ratios were shown. The results are represented as means ± standard error of the mean (SEM). Different letters indicate significant differences (P<.05).
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions