First published online March 12, 2004
doi: 10.1242/10.1242/jcs.00971
Journal of Cell Science 117, 1319-1328 (2004)
Published by The Company of Biologists 2004
Kisspeptin-10, a KiSS-1/metastin-derived decapeptide, is a physiological invasion inhibitor of primary human trophoblasts
Martin Bilban1,2,3,*,
Nassim Ghaffari-Tabrizi2,*,
Edith Hintermann1,
Sandra Bauer5,
Sylvia Molzer3,
Cristina Zoratti4,
Roland Malli4,
Andrew Sharabi1,
Ursula Hiden2,
Wolfgang Graier4,
Martin Knöfler5,
Fritz Andreae2,
Oswald Wagner3,
Vito Quaranta1 and
Gernot Desoye2,
1 Department of Cell Biology, The Scripps Research Institute, North Torrey Pines Road, La Jolla, 92037 CA, USA
2 Clinic of Obstetrics and Gynecology, Karl Franzens University Graz, Auenbruggerplatz 14, 8036 Graz, Austria
3 Institute for Laboratory Medicine, University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria
5 Clinic of Obstetrics and Gynecology, University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria
4 Institute for Medical Biochemistry, Karl-Franzens-University of Graz, Harrachgasse 21/III, 8010 Graz, Austria
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Fig. 1. KiSS-1 sequence and cleavage products resulting in various kisspeptins.
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Fig. 2. KiSS-1 and KiSS-1R are differentially expressed in FT and term human trophoblast cells. (A) RT-PCR of total RNA of freshly isolated FT or term trophoblasts (representative experiment: left panel). (B) Western blotting of FT and term trophoblasts. Lysates were probed with anti-Kp145/Kp-54 antiserum. With purified Kp-54 (5 ng) the antiserum produced a single band at 5-6 kDa, which is in good agreement with the theoretical MW for Kp-54 (5858.0 kDa; lane `M'). The antibody reacted only with lysates from FT trophoblasts producing one prominent band at  15-16 kDa, which is in good agreement with the calculated Mr for Kp-145 (15.391 kDa). ß-actin served as loading control. (C) FT trophoblast conditioned media were subjected to MALDI-TOF analysis after reverse phase-HPLC fractionation. The theoretical masses for C-terminally amidated Kps-10, -13, -14 and -54 are 1304, 1626, 1704 and 5858, respectively. Kp-54 was identified also as Na-adduct (m/z: 5876). The masses at around 2600 are unidentified.
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Fig. 3. Kisspeptin-10 raises intracellular Ca2+ in isolated first trimester human trophoblasts. FT trophoblasts were stimulated with different Kps and intracellular [Ca2+] was measured (A). Only Kp-10 resulted in an increase in intracellular [Ca2+] (n=9), whereas Kp-13, -14 and -54 were ineffective (n=6). (B) Concentration-response curve for Kp-10 on intracellular free Ca2+ concentration in isolated FT trophoblasts. The intracellular [Ca2+] is expressed as ratio (F340/F380) (mean±s.e.m.; n=6-9). The EC50 was found to be 21 (16-29) nM (95% confidential interval).
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Fig. 4. KiSS-1/Kp-54 and KiSS-1R localization in first trimester placenta. Sections of human FT placental tissue at week 6-10 of gestation showing the localization of KiSS-1/Kp-54 (A,B) and KiSS-1R (E,F). KiSS-1 and KiSS-1R mRNA were detected by in situ hybridization as dark blue precipitates, whereas their respective proteins were detected using affinity purified polyclonal antibodies evident as dark red precipitate. Sense probes (C,G) and nonimmune sera (D,H) produced no detectable signal. KiSS-1 mRNA (A) and Kp-145/Kp-54 protein (B) were detected mainly on the outer (syncytiotrophoblast) surface of villi. Higher magnification showed that KiSS-1/Kp-54 expression is restricted to the syncytiotrophoblast (insets; A and B; bars, 25 µm), whereas it was undetectable in villous (vCT) and extravillous (evCT) cytotrophoblasts. KiSS-1R mRNA is located in the syncytiotrophoblast (ST), villous (vCT) and extravillous (evCT) cytotrophoblasts (E). This mRNA staining pattern is paralleled by that of KiSS-1R protein (F). Bars, 50 µm.
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Fig. 7. KiSS-1 and KiSS-1R mRNA and protein expression in first trimester placenta. The histology of the maternal-fetal interface and KiSS-1 (A) and KiSS-1R (B) expression patterns are shown schematically from in situ hybridization and immunohistochemistry staining (Fig. 4). AV, anchoring villus; evCT: extravillous cytotrophoblast; ST, syncytiotrophoblast; vCT, villous cytotrophoblast.
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Fig. 5. Kisspeptin-10 inhibits trophoblast outgrowth and migration, but not proliferation in first trimester human villous explant cultures. Villous explants from 6-9 weeks' gestation were maintained in culture for 72 hours in the absence (A,B) or presence of 0.3 µM (C,D) or 1.0 µM (E,F) Kp-10. Identical villi were photographed at 48 or 72 hours. The dark areas are tissue, and sheets of outgrowing cytotrophoblast can readily be observed in the untreated cultures (A,B; arrows mark the limits/boundaries of the outgrowth). Kp-10 treatment profoundly decreases trophoblast outgrowth from the distal end of the villous tips when compared with control villous explants. Magnification: x40. (G-J) Proliferative potential of placental villi was determined by incorporation of BrdU after 24 hours in culture in the absence (G,I) or presence (H,J) of 1 µM Kp-10. Villus sections were stained with an anti-BrdU antibody, which detects cells in the S-phase (G,H), or were labeled with the nuclear stain dapi (I,J). The nonproliferating syncytiotrophoblast (ST) is devoid of anti-BrdU staining (G,H). Cell column (CC) formation by villus explants maintained in the absence or presence of Kp-10 (0.3 µM) was similar. In addition, no significant difference in the proportion of nuclei that incorporated BrdU was detected. Six villi per treatment were examined per placenta and the experiment was repeated three times. Magnification: x400. The extent of migration was increased between 48 and 72 hours under all conditions. Migration distance was reduced already at 0.3 µM Kp-10 at both 48 and 72 hours (K). The higher concentration of 1 µM did not augment the effect. *P<0.05 vs control. VS, villous stroma.
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Fig. 6. Kisspeptin-10 inhibits migration and gelatinolytic activity, but not proliferation of isolated first trimester human trophoblasts. (A) Microscopic image of the abluminal side of collagen I (Col I)- or fibronectin (FN)-coated membranes after 48 hours during which isolated FT trophoblasts (1x105 per filter, week 6-9 of gestation) migrated across the membrane in the absence or presence of 0.5 µM Kp-10. (B) Bars on left: Addition of Kp-10 (0.5 µM, black bars) reduced the number of cells that migrated through the filter pores. Results are expressed as mean±s.e.m. (n=3 different trophoblast isolations) of cell migration relative to unstimulated cells set as 1 (white bars) (*P<0.05 vs control). Bars on right: To assess cell proliferation, 7x104 freshly isolated FT trophoblasts were plated on collagen I in the absence (white bar) or presence of Kp-10 (black bars) added at the time of seeding. Cell numbers were counted after 48 hours. Results are presented as cell number (mean±s.e.m.; n=3) expressed relative to control (=100%). Kp-10 did not affect FT trophoblast proliferation significantly. ns, not significant vs control. (C) Conditioned medium from isolated FT trophoblasts plated on collagen I for 24 or 48 hours was subjected to gelatin zymography. Proteolytic activity was noted for the 72-kDa collagenase corresponding to pro-MMP-2. Protein molceular weight markers are indicated on the right. (D) Addition of Kp-10 suppressed 72-kDa collagenase (MMP-2) activity (*P<0.01 vs control).
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© The Company of Biologists Ltd 2004
