Kisspeptin stimulates progesterone secretion via the Erk1/2 mitogen-activated protein kinase signaling pathway in rat luteal cells

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1 Kisspeptin stimulates progesterone secretion via the Erk1/2 mitogen-activated protein kinase signaling pathway in rat luteal cells Jing Peng, M.D., a Min Tang, M.D., b Bao-Ping Zhang, M.D., a Peng Zhang, B.S., a Ting Zhong, M.D., a Teng Zong, B.S., a Bei Yang, M.D., a and Hai-Bin Kuang, M.D., Ph.D. a a Department of Physiology and b Department of Cell Biology, School of Medicine, Nanchang University, Nanchang, Jiangxi, People's Republic of China Objective: To observe the effect of kisspeptin on the endocrine function of rat luteal cells. Design: Experimental animal study. Setting: Research institute laboratory. Animal(s): Immature Sprague-Dawley rats. Intervention(s): The expression of kisspeptin and its receptor, GPR54, in immature rat ovaries treated with gonadotropin was observed via immunohistochemistry and real-time polymerase chain reaction. Then recombinant kisspeptin was used to examine the effect on the endocrine function of rat luteal cells. Main Outcome Measure(s): Expression and localization of kisspeptin, localization of GPR54, P and E 2 secretion, expression of steroidogenic enzymes, and phosphorylation of Erk1/2. Result(s): Real-time polymerase chain reaction indicated that ovarian KiSS-1 mrna levels increased significantly, showing a peak at the luteal period in gonadotropin-primed immature rats. Immunostaining analysis showed that after gonadotropin treatment, kisspeptin was strongly localized in theca cells, the interstitial compartment, and the corpus luteum and that GPR54 protein was clearly detected in the corpus luteum of rat ovaries. In cultured luteal cells, kisspeptin treatment augmented basal and hcg-induced P levels but not E 2 production, with concomitant increases detected in the transcript levels of key steroidogenic enzymes (StAR, CYP11A, and 3b-HSD). Furthermore, treatment with kisspeptin increased the phosphorylation of Erk1/2 mitogen-activated protein kinase in cultured luteal cells. Conclusion(s): The kisspeptin/gpr54 signaling system could stimulate P secretion in rat luteal cells via the Erk1/2 mitogen-activated protein kinase signaling pathway, suggesting an important role for the function of the corpus luteum. (Fertil Steril Ò 2013;99: Ó2013 by American Society for Reproductive Medicine.) Key Words: Kisspeptin, GPR54, progesterone, luteal cell, corpus luteum Discuss: You can discuss this article with its authors and with other ASRM members at fertstertforum.com/pengj-kisspeptin-progesterone-gpr54-luteal-cell/ Use your smartphone to scan this QR code and connect to the discussion forum for this article now.* * Download a free QR code scanner by searching for QR scanner in your smartphone s app store or app marketplace. Kisspeptin, a neuropeptide hormone encoded by the KiSS-1 gene, and its receptor, GPR54, have recently been recognized as fundamental activators of the gonadotropic axis with key roles in the Received July 13, 2012; revised December 3, 2012; accepted December 5, 2012; published online January 8, J.P. has nothing to disclose. M.T. has nothing to disclose. B.-P.Z. has nothing to disclose. P.Z. has nothing to disclose. T.Z. has nothing to disclose. T.Z. has nothing to disclose. B.Y. has nothing to disclose. H.-B.K. has nothing to disclose. The first two authors contributed equally to this work. This work was supported by the National Natural Science Foundation of China (grant nos , , , and ) and the Foundation of the Education Department of Jiangxi Province (grant no. GJJ12071). Reprint requests: Hai-Bin Kuang, M.D., Ph.D., Professor, Department of Physiology, School of Medicine, Nanchang University, Nanchang, Jiangxi , People's Republic of China ( hb. kuang@gmail.com). Fertility and Sterility Vol. 99, No. 5, April /$36.00 Copyright 2013 American Society for Reproductive Medicine, Published by Elsevier Inc. control of gonadotropin secretion and the onset of puberty (1, 2). In 1996, KiSS-1 mrna was first found in melanoma cell lines with different metastatic capacities using the subtractive hybridization method (3). However, it was not until 2001 that the peptide products of KiSS-1 were identified, including kisspeptin-54 (also known as metastin), kisspeptin-14, kisspeptin- 13, and kisspeptin-10. These peptides are derived from the differential proteolytic processing of a 145 amino acid precursor and globally referred to as kisspeptins, which all harbor an Arg VOL. 99 NO. 5 / APRIL 2013

2 Fertility and Sterility Phe-NH motif at the C-terminus, distinguishing them from the RF-amid peptide superfamily (4 6). In 2003, two research groups independently reported that deletion of and inactivating mutations in GPR54 were associated with idiopathic hypogonadotropic hypogonadism characterized by low gonadotropin and sex steroid levels in humans and mice (7, 8). Subsequent physiological and pharmacological studies demonstrated that kisspeptins primarily act at the hypothalamus, where they operate as essential activators of GnRH function and stimulate FSH and especially LH secretion (2, 9). Although central kisspeptin has been proven to be an essential element in the hypothalamic-pituitary-gonadal axis, additional potential actions of kisspeptins at other levels of the gonadotropic axis (for example, direct action on gonadal tissues) cannot be ruled out (10 12). In fact, KiSS-1 protein has been detected in human and rat ovarian tissue and cultured granulosa-lutein cells (11, 13), and the levels of the rat ovarian KiSS-1 gene have been shown to fiuctuate in an estrous cycle dependent manner (14). Furthermore, GPR54 protein was also found in human and rat theca and luteal cells and in fish ovaries (12, 14, 15). These data suggested a potential role for kisspeptins in the local control of ovarian function. On the other hand, in the human placenta, KiSS-1 protein is highly expressed in the syncytiotrophoblast adjacent to the maternal blood, and plasma kisspeptin levels increase dramatically in the first trimester of pregnancy (12, 16), leading to the intriguing possibility that kisspeptin might act in a manner similar to hcg, which sustains luteal development and stimulates P secretion (17). Furthermore, kisspeptin can increase aldosterone production in both fetal neocortex adrenal cells and H295R adrenal cells (18). Based on all of this evidence, we hypothesized that kisspeptins might be involved in the regulation of ovarian luteal function. In the present study, we observed the expression and localization of kisspeptin and its receptor, GPR54, via real-time polymerase chain reaction (PCR) and immunohistochemistry in immature rat ovaries treated with gonadotropin. We also investigated the effect and mechanism of the action of recombinant kisspeptin regarding the endocrine function of rat luteal cells in vitro. MATERIALS AND METHODS Animals and Treatments Immature Sprague-Dawley (22- to 24-day-old) rats were housed under a constant photoperiod (14 hours light: 10 hours dark cycle) and humidity, with free access to standard rat food and water. The experimental protocol was reviewed and approved by the Committee for Ethics in Animal Experiments of the Nanchang University School of Medicine. After being housed for 1 day, the rats received an SC injection of 50 IU of pregnant mare's serum gonadotropin (PMSG) (NingBo Biological Technology) to induce follicular maturation. Fortyeight hours later, they were administered 50 IU of hcg (NingBo Biological Technology) to induce ovulation. The ovaries were collected for analysis at 24 and 48 hours after PMSG injection and at 3 hours and 7 days after hcg treatment. Isolation and Culture of Rat Luteal Cells The isolation of cells from luteinized ovaries was performed as described elsewhere with one slight modification (19). First, immature rats received an SC injection of 50 IU of PMSG, and 48 hours later, they were administered 50 IU of hcg. The rats were euthanized under ether anesthesia 7 days after hcg injection. After the removal of fat and capsule tissue from the ovary, the corpora lutea were minced in DMEM/ F12 medium and digested with 0.1% collagenase (type I; Sigma-Aldrich) and 15 IU/mL DNase I (Sigma-Aldrich) for 60 minutes at 37 C in a shaking water bath. The digested suspension was filtered through 75 and 38 mm strainers to remove debris, and the filtrate was centrifuged at 500 g for 10 minutes. The obtained cell pellets were washed twice with the medium. Cell viability was tested and was found to be greater than 91.5% using the trypan blue exclusion method. The number of cells was subsequently adjusted to viable cells/ml, which were then cultured in 48-well plates with DMEM/F12 containing 100 IU/mlLpenicillin and 100 mg/ml streptomycin in 95% air and 5% CO 2. Luteal cells were first treated with various concentrations of hcg (BioVision). It was determined that hcg at a concentration of 4 IU/ ml has an optimal effect regarding P and E 2 secretion. Then the luteal cells were treated with recombinant kisspeptin-10 (Phoenix Pharmaceuticals Inc.) with or without 4 IU/mL hcg. The culture media were collected after 24 hours of incubation and stored at 80 C until P and E 2 determination was performed. RNA Isolation and Real-Time PCR Total RNA was extracted from ovary tissues or luteal cells with the TRIzol reagent (Invitrogen) according to the manufacturer's protocol. RNA concentrations and purity were determined using a spectrophotometer (Shimadzu). Based on the absorbance at 260 nm, the concentration of the RNA samples was adjusted to 1 mg/ml before performing reverse transcription in a 25-mL reaction mixture (Promega). Real-time PCR was then performed in a 20-mL reaction volume containing 10 mlof2brilliant SYBR Green QPCR Master Mix (Tiangen Biotech), 2 ml of template cdna, 0.5 mm of primers, and 300 nm of reference dye using the ABI thermal cycler The thermal cycling conditions were 95 C for 15 minutes, followed by 40 cycles at 94 C for 15 seconds, 57 C for 30 seconds, and 72 C for 30 seconds. Melting curve analysis and agarose gel electrophoresis were conducted after the realtime PCR assays to monitor PCR product purity. The realtime PCR results were analyzed using ABI Prism 7500 SDS software (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed for normalization. The following primers were used: GAPDH, sense, 5 0 -CTC ATG ACC ACA GTC CAT GC-3 0, antisense, TTC AGC TCT GGG ATG ACC TT-3 0 ; StAR, sense, 5 0 -AGA TGA AGT GCT AAG TAA GGT GGT G-3 0, antisense, 5 0 -CCA GTT CTT CAT AGA GTC TGT CCA T-3 0 ;3b-HSD, sense, 5 0 -AGA CCA TCC TAG ATG TCA ATC TGA A-3 0, antisense, 5 0 -CAG GAT GAT CTT CTT GTA GGA GT-3 0 ; CYP11A, sense, 5 0 -CCA AGT TCA ACC TCA TCC TGA-3 0, antisense, 5 0 -CGT GTG ACT GCA GCC TGC AA-3 0 ; and KiSS-1, sense, 5 0 -TGG CAC CTG TGG VOL. 99 NO. 5 / APRIL

3 ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY TGA ACC CTG AAC-3 0, antisense, 5 0 -ATC AGG CGA CTG CGG GTG GCA CAC-3 0. Immunohistochemistry Tissues were fixed in 4% paraformaldehyde. Tissue sections (5 mm) were deparaffinized in xylene and hydrated in a graded series of ethanol solutions, followed by water. Endogenous peroxidase activity was blocked by incubating the sections in 3% hydrogen peroxide for 10 minutes. Nonspecific binding was blocked in 5% bovine serum albumin for 60 minutes. Then the sections were incubated in rabbit anti-kisspeptin (1:150, Millipore) or rabbit anti-gpr54 (1:300, Abcam) overnight at 4 C. After washing in phosphate-buffered saline (PBS), the sections were incubated with a secondary antibody for 1 hour at room temperature. Detection was then carried out in the sections using fresh diaminobenzidine solution, together with counterstaining with Harris's hematoxylin. In some sections, the primary antibodies were replaced with rabbit preimmune IgG as a negative control. Western Blot Analysis To examine intracellular signaling, luteal cells treated with recombinant kisspeptin and/or hcg were washed twice with PBS and incubated with RIPA lysis buffer supplemented with a phosphatase inhibitor cocktail (Applygen Technologies) and phenylmethanesulfonyl fluoride (PMSF). Cell lysates were then centrifuged, and the supernatants were separated to be used in immunoblotting analyses to detect Erk1/2, phospho-erk1/2, P38, phosphor-p38, Akt, and phosphor-akt. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Applygen Technologies). The following antibodies were used in these analyses: anti-beta-actin (1:500), anti-p38 MAP Kinase, anti-phospho-p38 MAP Kinase, anti-p44/42 MAP Kinase, and anti-phospho-p44/42 MAP kinase (1:1,000 dilution; Cell Signaling Technology). The nitrocellulose membranes were then incubated with horseradish peroxidase conjugated secondary antibodies and visualized via enhanced chemiluminescence (Pierce). A minimum of three blots from different samples were performed, and the band intensities were measured with the Quantity One program (Bio-Rad). The data were corrected for background and normalized to b-actin expression. Hormone Measurements After cell culturing and treatment, the supernatants of the cell media were collected and stored at 80 C to be used for hormone measurements. P 4 and E 2 were measured using 125 I-labeled radioimmunoassay (RIA) kits (Jiuding Medicine Biotechnology). The intra- and interassay coefficients of variation when using these kits did not exceed 10%. The crossreactivities with other peptides and steroid hormones in these kits did not exceed 4%. The detection limits of the P 4 and E 2 kits are 0.25 ng/ml and 2 pg/ml, respectively. Statistical Analysis All data were presented as the means SE. The results were analyzed using one or two-way analysis of variance (ANOVA), followed by an least significant difference (LSD) post hoc test, or using Student's t test between two groups, and P<.05 was considered statistically significant. All statistical analyses were performed in the Statistical Package for the Social Sciences (SPSS) RESULTS Gonadotropin Treatment Stimulates KiSS-1 Expression in Rat Ovaries Previously published data suggested that KiSS-1 expression was regulated by gonadotropin (14). In the present investigation, the expression profiles of KiSS-1 mrna and protein were thoroughly examined in immature rat ovaries after gonadotropin treatment. Real-time PCR results indicated that the ovarian KiSS-1 mrna level significantly increased after PMSG and hcg treatment, showing a peak at 7 days after hcg treatment (Fig. 1A). To further investigate the cell types expressing kisspeptin, we analyzed the distribution pattern of KiSS-1 protein in rat ovaries after gonadotropin treatment using immunohistochemistry (Fig. 1B). In immature rat ovaries, the KiSS-1 protein showed a weak staining signal in the granulosa cells of primary follicles (Fig. 1B). At 24 and 48 hours after PMSG and 3 hours after hcg treatment, stronger signals were detected in the theca cells and interstitial compartment of the rat ovary, whereas weak staining remained in the granulosa cells (Fig. 1B). At 7 days after hcg treatment of PMSG-primed rats, strong signals were mainly found in the corpora lutea. In contrast, the granulosa cells in growing follicles showed no staining (Fig. 1B). Localization of GPR54 in Rat Ovaries after Gonadotropin Treatment Because kisspeptins were dominantly expressed in the corpora lutea, GPR54 immunostaining was selectively conducted in a subset of ovarian samples, consisting of ovaries treated with vehicle alone and 7 days after hcg treatment of PMSG-primed rats. In immature rat ovaries treated with vehicle alone, the granulosa cell layer of primary follicles showed clear immunostaining for GPR54, whereas the interstitial compartment was negative for GPR54 staining (Fig. 1C). At 7 days after hcg injection, GPR54 immunostaining was strongly detected in the corpus luteum (Fig. 1C). However, no immunostaining was observed in the interstitial compartment or negative control (Fig. 1C). Dose-Dependent Effects of Kisspeptin on Steroidogenesis in Cultured Luteal Cells To study the possible effects of kisspeptin on steroidogenesis, we isolated luteal cells from immature rat ovaries 7 days after hcg treatment, and luteal cells were treated with hcg and/or recombinant kisspeptin. As shown in Figure 2A and B, P and E 2 secretion was stimulated in a dose-dependent manner by hcg, and 8 IU/mL hcg maximized this response. For subsequent experiments, a dose of 4 IU/mL hcg was chosen for use. When cells were treated with increasing doses of kisspeptin alone, a significant increase in P production was observed, 1438 VOL. 99 NO. 5 / APRIL 2013

4 Fertility and Sterility 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 ¼ 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 mm. O ¼ oocyte; F ¼ follicle; G ¼ granulosa cells; T ¼ thecal cells; I ¼ interstitial compartment; CL ¼ corpus luteum. with a maximal increase observed at M(Fig. 2C). Combined treatment with kisspeptin and hcg was able stimulate P production more strongly than kisspeptin or hcg alone (P<.05; Fig. 2C). Although kisspeptin augmented basal and hcg-induced P production, no significant changes in E 2 production were detected in these culture media (P>.05; Fig. 2D). Effect of Kisspeptin on Steroidogenic Enzymes in Luteal Cells To investigate the mechanism underlying the enhancement of P production by kisspeptin, we detected the mrna levels of three key steroidogenic enzymes: steroidogenic acute regulatory protein (StAR; Fig. 3A), cytochrome P45011A (CYP11A; Fig. 3B), and 3-hydroxysteroid dehydrogenase (3-bHSD; Fig. 3C), via real-time PCR. Based on the dosedependency of the P stimulation induced by kisspeptin and hcg, luteal cells were incubated with 4 IU/mL hcg and/or M kisspeptin for 24 hours. Compared with the control group, real-time PCR results indicated that treatment with kisspeptin either alone or together with hcg significantly augmented the levels of StAR (2.62- and 3.92-fold, respectively; P<.05) and CYP11A mrna (2.59- and 3.90-fold, respectively; P<.05). However, treatment with kisspeptin alone had no significant effect on the level of 3-bHSD mrna (P>.05), while the combination of kisspeptin and hcg significantly enhanced the transcript levels of 3-bHSD (P<.05; Fig. 3C). VOL. 99 NO. 5 / APRIL

5 ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY FIGURE 2 Dose-dependent effect of kisspeptin on basal and hcg-induced P and E 2 secretion in cultured luteal cells. (A) P content in the culture media of luteal cells treated with different doses of hcg. (B) E 2 content in the culture media of luteal cells treated with different doses of hcg. (C) Basal and hcginduced (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) E 2 content in the culture media of luteal cells treated with different doses of kisspeptin. The results are represented as the mean SE (n ¼ 4) from one of three independent experiments. Groups with different superscript letters are significantly different (P<.05, ANOVA followed by LSD multiple range test). Kisspeptin Phosphorylates Erk1/2 in Cultured Luteal Cells Next we aimed to determine the signaling pathways activated by kisspeptin in cultured luteal cells. In diverse tissues and cells, kisspeptin has been found to activate different MAPK components and the PI3K/Akt pathway (12, 20). Here we examined two well-characterized subfamilies of the MAPK system (P44/42 MAPK [Erk1/2] and P38 MAPK) and the Akt pathway. The isolated luteal cells were treated with increasing doses of kisspeptin with or without 4 IU/mL hcg for 60 minutes before immunoblotting analysis. As shown in Figure 4, treatment with kisspeptin at a concentration of M stimulated Erk1/2 activation. In addition, when cells were cotreated with kisspeptin and hcg, the phosphorylation of Erk1/ 2 was further increased compared with the group treated with hcg alone (Fig. 4A and B). However, treatment with kisspeptin alone did not affect P38 MAPK and Akt phosphorylation under the same experimental conditions (data not shown). Furthermore, RIA analysis indicated that PD98059 (an Erk-specific inhibitor) significantly attenuated kisspeptinmediated P production (P<.01), but LY (Akt inhibitor) did not significantly alter P production (P>.05; Fig. 4C). DISCUSSION The current study demonstrated that ovarian KiSS-1 mrna levels significantly increased in gonadotropin-primed immature rats, showing a peak at the luteal period. Immunohistochemical results indicated that after gonadotropin treatment, kisspeptin was strongly expressed in the theca cells, interstitial compartment, and corpus luteum and that GPR54 protein was strongly localized to the corpus luteum of rat ovaries. In cultured luteal cells, recombinant kisspeptin significantly augmented basal and hcg-induced P production, but not E 2 secretion, likely mediated via stimulation of key steroidogenic enzymes, including CYP11A, StAR, and 3-bHSD. In addition, Western blot analysis showed that treatment with kisspeptin significantly increased the phosphorylation of Erk1/2 mitogen-activated protein kinase (MAPK) in cultured luteal cells. Although compelling evidence has demonstrated that the components of the KiSS-1/GPR54 system are expressed and act primarily at the central hypothalamic level, some data have indicated that the KiSS-1 and/or GPR54 gene is expressed in different peripheral tissues, including ovary, oviduct, placenta, and other nonreproductive organs, such as the pancreas (6, 11, 13, 14, 21, 22). On the basis of the profile of KiSS-1/GPR54 in the rat ovary (11, 14), in the present study, we first provided a comprehensive overview of the expression pattern of kisspeptins and GPR54 in immature rat ovaries after gonadotropin treatment. The results indicated that ovarian KiSS-1 mrna levels exhibited a significant elevation after gonadotropin treatment, showing a peak 7 days after hcg treatment. These findings are not 1440 VOL. 99 NO. 5 / APRIL 2013

6 Fertility and Sterility 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 M kisspeptin for 24 hours. The expression levels of (A) StAR, (B) CYP11A, and (C) 3b-HSD mrna were quantified using real-time PCR and normalized to GAPDH levels. The results are represented as the mean SE (n ¼ 3). Groups with different superscript letters are significantly different (P<.05, ANOVA followed by LSD multiple range test). completely consistent with Castellano et al.'s results (14), in which the maximal magnitude occurred 3 hours after combined PMSG and hcg treatment. The difference between these results could have been due to the different sampling times and assay methods used in these studies. In Castellano et al.'s study, samples were collected at only one time point, while in our study, multiple samples were collected at different time intervals (14). On the other hand, immunoblotting analysis indicated that GPR54 protein levels presented no significant differences between rat ovaries treated with vehicle and hcg (Supplemental Fig. 1), which coincides with the levels of GPR54 mrna detected during the estrous cycle (14). Immunohistochemical results indicated that kisspeptin signals were strongly localized in the theca cells, interstitial compartment, and corpus luteum, and GPR54 protein was clearly localized in the corpus luteum of rat ovaries after gonadotropin treatment, similar to the results of previous immunohistochemical analyses (11, 14). Based on the pattern of GPR54 cellular distribution mostly localized in the corpus luteum (11, 14) and plasma kisspeptin levels dramatically augmented during pregnancy (16), there is hypothesized a potential role for kisspeptin in the modulation of the corpus luteum. In the present study, our data indicated that in cultured luteal cells, treatment with kisspeptin increased basal and hcg-induced P secretion but not E 2 secretion. This function could promote corpus luteum formation and the sustain development of a pregnancy. Associated with the essential role of luteal P in pregnancy, a complex series of factors (including luteotropic and luteolytic factors) have been demonstrated to be involved in regulating corpus luteum formation and activation (17, 23). Uterine prostaglandin F2a, endothelin-1, tumor necrosis factor-a, and monocyte chemotactic protein-1 have been postulated as luteinization inhibitors owing to their P-suppressing functions (17). On the other hand, some factors exhibit a luteotropic function, including PRL- or LH-like hormones, hcg, steroids, and peptide and arachidonate metabolites (24). Among the stimulators of luteinization, our data have demonstrated that kisspeptin is similar to hcg, which is secreted by trophoblast cells of the developing placenta, and promotes P biosynthesis and luteinization of the ovary (16, 17, 25). To further explore the molecular mechanisms underlying the action of kisspeptin in cultured luteal cells, we analyzed components of the signaling pathways mediated by PI3K/ Akt and MAPKs, including well-characterized ERK1/2 and VOL. 99 NO. 5 / APRIL

7 ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY FIGURE 4 steroidogenic enzymes and phosphorylate Erk1/2 MAPK in cultured luteal cells. The results suggest that the local KiSS- 1/GPR54 signaling system could play a paracrine/autocrine role in the regulation of corpus luteum function. Acknowledgments: The authors thank Dr. Hong Xu of the department of Physiology, School of Medicine, Nanchang University, for proofreading this manuscript. 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 and LY ) on P secretion in cultured luteal cells. The results are represented as the mean SE (n ¼ 4). Groups with different superscript letters are significantly different (P<.01, ANOVA followed by LSD multiple range test). p38 MAPK proteins (20). In Western blot analyses, we found that kisspeptin treatment significantly increased basal and hcg-stimulated phosphorylation of ERK1/2 after 60 minutes in luteal cells. This result is consistent with those initially reported for KiSS-1/GPR54 signaling using different cell models, such as mhypoe-38 cells and GT1-7 and GN11 GnRH neuronal cells (6, 20). Our data further demonstrated a potential difference in KiSS-1/GPR54 signaling in cultured luteal cells. Kisspeptin did not significantly alter the phosphorylation of Akt or p38, whereas it has been shown to stimulate the phosphorylation of Akt and p38 in a variety of other cell types, including GPR54-transfected Chinese hamster ovary cells (6) and trophoblast cells (26). These different findings could result from different cell specificities. In conclusion, our data provide the first evidence that kisspeptin could augment P secretion via stimulating key REFERENCES 1. Roa J, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M. New frontiers in kisspeptin/gpr54 physiology as fundamental gatekeepers of reproductive function. Front Neuroendocrinol 2008;29: Roseweir AK, Millar RP. The role of kisspeptin in the control of gonadotrophin secretion. Hum Reprod Update 2009;15: Lee JH, Miele ME, Hicks DJ, Phillips KK, Trent JM, Weissman BE, et al. KiSS-1, a novel human malignant melanoma metastasis-suppressor gene. 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Expression of KiSS-1 in rat ovary: putative local regulator of ovulation? Endocrinology 2006;147: Filby AL, Aerle R, Duitman JW, Tyler CR. The kisspeptin/gonadotropinreleasing hormone pathway and molecular signaling of puberty in fish. Biol Reprod 2008;78: Horikoshi Y, Matsumoto H, Takatsu Y, Ohtaki T, Kitada C, Usuki S, et al. Dramatic elevation of plasma metastin concentrations in human pregnancy: metastin as a novel placenta-derived hormone in humans. J Clin Endocrinol Metab 2003;88: Niswender GD, Juengel JL, Silva PJ, Rollyson MK, McIntush EW. Mechanisms controlling the function and life span of the corpus luteum. Physiol Rev 2000;80: Nakamura Y, Aoki S. Metastin stimulates aldosterone synthesis in human adrenal cells. Reprod Sci 2007;14: Sugino N, Takiguchi S, Kashida S, Takayama H, Yamagata Y, Nakamura Y, et al. Suppression of intracellular superoxide dismutase activity by antisense 1442 VOL. 99 NO. 5 / APRIL 2013

8 Fertility and Sterility oligonucleotides causes inhibition of progesterone production by rat luteal cells. Biol Reprod 1999;61: Kim GL, Dhillon SS, Belsham DD. Kisspeptin directly regulates neuropeptide Y synthesis and secretion via the ERK1/2 and p38 mitogen-activated protein kinase signaling pathways in NPY-secreting hypothalamic neurons. Endocrinology 2010;151: Gaytan M, Castellano JM, Roa J, Sanchez-Criado JE, Tena-Sempere M, Gaytan F. Expression of KiSS-1 in rat oviduct: possible involvement in prevention of ectopic implantation? Cell Tissue Res 2007;329: Terao Y, Kumano S, Takatsu Y, Hattori M, Nishimura A, Ohtaki T, et al. Expression of KiSS-1, a metastasis suppressor gene, in trophoblast giant cells of the rat placenta. Biochim Biophys Acta 2004;1678: Stocco C, Telleria C, Gibori G. The molecular control of corpus luteum formation, function, and regression. Endocr Rev 2007;28: Hahlin M, Dennefors B, Johanson C, Hamberger L. Luteotropic effects of prostaglandin E2 on the human corpus luteum of the menstrual cycle and early pregnancy. J Clin Endocrinol Metab 1988;66: Piotrowska KK, Woclawek-Potocka I, Bah MM, Piskula MK, Pilawski W, Bober A, et al. Phytoestrogens and their metabolites inhibit the sensitivity of the bovine corpus luteum to luteotropic factors. J Reprod Dev 2006;52: Bilban M, Ghaffari-Tabrizi N, Hintermann E, Bauer S, Molzer S, Zoratti C, et al. Kisspeptin-10, a KiSS-1/metastin-derived decapeptide, is a physiological invasion inhibitor of primary human trophoblasts. J Cell Sci 2004;117: VOL. 99 NO. 5 / APRIL

9 ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY 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 e1 VOL. 99 NO. 5 / APRIL 2013

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