Kisspeptin stimulates progesterone secretion via the Erk1/2 mitogen-activated protein kinase signaling pathway in rat luteal cells Jing Peng, M.D., Min.

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Kisspeptin stimulates progesterone secretion via the Erk1/2 mitogen-activated protein kinase signaling pathway in rat luteal cells Jing Peng, M.D., Min Tang, M.D., Bao-Ping Zhang, M.D., Peng Zhang, B.S., Ting Zhong, M.D., Teng Zong, B.S., Bei Yang, M.D., Hai-Bin Kuang, M.D., Ph.D. Fertility and Sterility Volume 99, Issue 5, Pages 1436-1443.e1 (April 2013) DOI: 10.1016/j.fertnstert.2012.12.008 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Expression of KiSS-1 and GPR54 in ovaries of immature rats treated with gonadotropin. (A) Levels of KiSS-1 mRNA expression in ovaries of immature rats treated with gonadotropin determined via real-time PCR. The data are presented as the mean ± SE (n = 3) from one of three independent experiments. *P<.05, **P<.01 compared with the vehicle control. (B) Immunohistochemical localization of KiSS-1 protein in ovaries of immature rats treated with gonadotropin. Ovaries were sampled at 24 hours (b) and 48 hours (c) after PMSG treatment and at 3 hours (d) and 7 days (e) after hCG injection. Age-matched female rats treated with vehicle alone served as controls (a). (f) Negative control (without primary antibody). (C) Immunohistochemical localization of GPR54 protein in ovaries treated with vehicle (g) or gonadotropin (7 days after hCG treatment) (h). Scale bar, 50 μm. O = oocyte; F = follicle; G = granulosa cells; T = thecal cells; I = interstitial compartment; CL = corpus luteum. Fertility and Sterility 2013 99, 1436-1443.e1DOI: (10.1016/j.fertnstert.2012.12.008) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Dose-dependent effect of kisspeptin on basal and hCG-induced P and E2 secretion in cultured luteal cells. (A) P content in the culture media of luteal cells treated with different doses of hCG. (B) E2 content in the culture media of luteal cells treated with different doses of hCG. (C) Basal and hCG-induced (hCG, 4 IU/mL) P content in the culture media of luteal cells treated with different doses of kisspeptin. (D) Basal and hCG-induced (hCG, 4 IU/mL) E2 content in the culture media of luteal cells treated with different doses of kisspeptin. The results are represented as the mean ± SE (n = 4) from one of three independent experiments. Groups with different superscript letters are significantly different (P<.05, ANOVA followed by LSD multiple range test). Fertility and Sterility 2013 99, 1436-1443.e1DOI: (10.1016/j.fertnstert.2012.12.008) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Enhancement effect of kisspeptin treatment on the expression of mRNA for different steroidogenic enzymes in cultured luteal cells. Isolated luteal cells were cultured with 4 IU/mL hCG and/or 4 × 10−8 M kisspeptin for 24 hours. The expression levels of (A) StAR, (B) CYP11A, and (C) 3β-HSD mRNA were quantified using real-time PCR and normalized to GAPDH levels. The results are represented as the mean ± SE (n = 3). Groups with different superscript letters are significantly different (P<.05, ANOVA followed by LSD multiple range test). Fertility and Sterility 2013 99, 1436-1443.e1DOI: (10.1016/j.fertnstert.2012.12.008) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Kisspeptin treatment enhances the phosphorylation of Erk1/2 in cultured luteal cells. (A) Representative immunoblotting results for phospho-Erk1/2 (upper lane), total Erk1/2 (middle lane), and actin protein (lower lane) are shown. (B) Densitometric analyses of phospho-Erk1/2 from three independent experiments. The results are shown relative to total Erk1/2 and expressed as the mean ± SE *P<.05, **P<.01 compared with controls (without hCG group) or groups treated with hCG alone (with hCG group). (C) Effects of pharmacological inhibitors (PD 98059 and LY 294002) on P secretion in cultured luteal cells. The results are represented as the mean ± SE (n = 4). Groups with different superscript letters are significantly different (P<.01, ANOVA followed by LSD multiple range test). Fertility and Sterility 2013 99, 1436-1443.e1DOI: (10.1016/j.fertnstert.2012.12.008) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Expression levels of GPR54 protein in ovaries from immature rats treated with gonadotropin. (A) Representative immunoblotting results of GPR54 are shown from immature rat ovaries treated with vehicle or gonadotropin. (B) Densitometric analyses of GPR54 from three independent experiments. The results are shown relative to Actin and expressed as the mean ± SE. Fertility and Sterility 2013 99, 1436-1443.e1DOI: (10.1016/j.fertnstert.2012.12.008) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions