1. Sphingosine-1-phosphate inhibits H2O2-induced granulosa cell apoptosis via the PI3K/Akt signaling pathway 
Tatsuo Nakahara, M.D., Akira Iwase, M.D., Ph.D., Tomoko Nakamura, M.D., Mika Kondo, M.D., Bayasula, M.D., Hiroharu Kobayashi, M.D., Sachiko Takikawa, M.D., Shuichi Manabe, M.D., Maki Goto, M.D., Tomomi Kotani, M.D., Fumitaka Kikkawa, M.D. 
Fertility and Sterility 
Volume 98, Issue 4, Pages e1 (October 2012)
DOI: /j.fertnstert
Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Induction of ovarian granulosa cell apoptosis by H2O2 and the effect of sphingosine-1-phosphate (S1P) on H2O2-induced apoptotic cell death. Human cultured granulosa cells were harvested for 24 hours and collected for a Hoechst nuclear morphology analysis and assessment for apoptosis levels by Western blotting. (A) Representative phase contrast photographs of granulosa cells after vehicle and H2O2 treatment. Granulosa cells were treated with 0.01, 0.1, or 1.0 mM H2O2 for 24 hours. (Scale bar: 500 μm.) Apoptosis levels of granulosa cells after vehicle and H2O2 treatment. Bars represent the percentages of apoptotic nuclei counted in each treatment group and are expressed as the mean ± standard error of the mean (n = 3 per group). *Statistically significant differences between the vehicle and H2O2-treated cells (P<.05). (B) Apoptosis levels assessed by Western blotting using antibodies recognizing cleaved caspase-3. (C) Representative images of Hoechst staining of granulosa cells. Arrows indicate the apoptotic nuclei, the chromatin of which appeared condensed and fragmented under fluorescence microscopy. (Scale bar: 100 μm.) (D) Representative phase contrast photographs of granulosa cells after vehicle, H2O2, and H2O2 + S1P treatment. (Scale bar: 500 μm.) Apoptosis levels of granulosa cells after vehicle, H2O2 and H2O2 + S1P treatment. Bars represent the percentages of apoptotic nuclei counted in each treatment group and are expressed as the mean ± standard error of the mean (n = 3 per group). *Statistically significant differences in comparison with the vehicle treatment group (P<.05). **Statistically significant differences in comparison with the vehicle treatment group (P<.1). (E) Apoptosis levels assessed by Western blotting using antibodies recognizing cleaved caspase-3.
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3. Figure 2
Human granulosa cells express sphingosine-1-phosphate (S1P) receptors and effect of S1P receptor antagonists on the antiapoptotic effect of S1P. (A) mRNA expression of S1P1–5 receptors by human luteinized granulosa cells. (B) Granulosa cells pretreated with vehicle or S1P receptor antagonist before H2O2 + S1P treatment. Bars represent the percentages of apoptotic nuclei counted in each treatment group and are expressed as the mean ± SEM (n = 3 per group). VPC = VPC23019 (antagonist of S1P1/3 receptor); W146 (antagonist of S1P1 receptor); CAY = CAY10444 (antagonist of S1P3 receptor). *Statistically significant differences in comparison with the H2O2 + S1P treatment group (P<.05). (C) Apoptosis levels assessed by Western blotting using antibodies recognizing cleaved caspase-3.
Fertility and Sterility  , e1DOI: ( /j.fertnstert )
Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
Sphingosine-1-phosphate (S1P) induces a time- and concentration-dependent increase in the phosphorylation of Akt and extracellular-regulated kinase (ERK) in human luteinized granulosa cells. Granulosa cells were exposed to 1 μM S1P or 10 μM S1P for different times (5 to 30 minutes). (A) Western blot analyses of phospho-Akt, total-Akt, phospho-ERK, total-ERK, and beta-actin. Western blot analyses shown in autoradiograms are representative of three independent experiments. The band intensities of phosphoproteins were normalized against (B) total-Akt or (C) or total-ERK, and are expressed as a fold increase in relation to the corresponding control. (D) Representative phase contrast photographs of granulosa cells after H2O2, H2O2 + S1P, H2O2 + S1P + LY (LY), and H2O2 + S1P + PD98059 (PD) treatment. (Scale bar: 500 μm.) Apoptosis levels of granulosa cells after H2O2, H2O2 + S1P, H2O2 + S1P + LY, and H2O2 + S1P + PD treatment. Bars represent the percentages of apoptotic nuclei counted in each treatment group and are expressed as the mean ± standard error of the mean (n = 3 per group). *Statistically significant differences in comparison with the H2O2 + S1P treatment group (P<.01). (E) Apoptosis levels assessed by Western blotting using antibodies recognizing cleaved caspase-3.
Fertility and Sterility  , e1DOI: ( /j.fertnstert )
Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

5. Figure 4
Effect of sphingosine-1-phosphate (S1P) receptor antagonists on S1P-induced Akt and extracellular-regulated kinase (ERK) phosphorylation in human luteinized granulosa cells. Granulosa cells were pretreated with S1P1 receptor antagonist W146, S1PR1/3 receptor antagonist VPC23019, and S1P3 receptor antagonist CAY10444 before stimulation with insulin-like growth factor 1 (IGF-1) or S1P for 10 minutes.
Fertility and Sterility  , e1DOI: ( /j.fertnstert )
Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

6. Supplemental Figure 1
Schema of the inhibitory mechanism of S1P in oxidative stress–induced granulosa cell apoptosis. Generally, oxidative stress induces mitochondrial dysfunction, consequent release of cytochrome c, and activation of caspase-3. The S1P ligands in follicular fluid may bind to and activate mainly S1PR1 and S1PR3 located in the granulosa cell membrane. Also S1P may inhibit oxidative stress–induced granulosa cell apoptosis by suppressing the release of caspase-3 mainly via the PI3K/Akt signaling pathway in the cytosol. The effect of IGF-1 (as a positive control) on ERK and PI3K/Akt activation is also shown. GPCR = G protein-coupled receptor; RTK = receptor tyrosine kinase.
Fertility and Sterility  , e1DOI: ( /j.fertnstert )
Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions