Expression of leucine-rich repeat-containing G-protein-coupled receptors in the human cyclic endometrium  Claudia A. Krusche, Ph.D., Tina Kroll, Henning M. Beier, M.D., Ph.D., Irmgard Classen-Linke, Ph.D.  Fertility and Sterility  Volume 87, Issue 6, Pages 1428-1437 (June 2007) DOI: 10.1016/j.fertnstert.2006.11.061 Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 The obtained products (LGR-4, LGR-5, LGR-7, and the housekeeping gene 5-ALAS), the amplification curves of typical endometrial samples, and the negative controls obtained in the LightCycler runs are shown. The insert in the LGR-4 run shows a run that was stopped at 35 cycles. At that point of the PCR reaction, no product was generated in the −RT control. Krusche. LGR-4, -5, and -7 mRNA in human endometrium. Fertil Steril 2007. Fertility and Sterility 2007 87, 1428-1437DOI: (10.1016/j.fertnstert.2006.11.061) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Endometrial mRNA expression of LGR-4 (A), LGR-5 (B), and LGR-7 (C) quantified by real time RT-PCR (n = 26) and assessment of the mRNA size by Northern blot in representative samples. Krusche. LGR-4, -5, and -7 mRNA in human endometrium. Fertil Steril 2007. Fertility and Sterility 2007 87, 1428-1437DOI: (10.1016/j.fertnstert.2006.11.061) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Representative results of LGR-4, LGR-5, and LGR-7 mRNA expression in endometrial cell types directly after isolation from hysterectomy material in the left column. epi = epithelial cells; fib = fibroblasts. The LGLs are derived from an endometrial sample from day 24 of the cycle. In the right column, representative LGR-4, LGR-5, and LGR-7 mRNA expression in endometrial epithelial cells and fibroblasts after in vitro cultivation using different hormone treatments (0 = without steroids) are depicted. The mRNA expression of the housekeeping gene Ribosomal protein S26 served as a control. −RT1 = RNA from cells directly after isolation from hysterectomy material; −RT2 = RNA from in vitro cultured endometrial cells; w = water. Since the LGR-5 primer pair is intron spanning, no −RT control was performed. Krusche. LGR-4, -5, and -7 mRNA in human endometrium. Fertil Steril 2007. Fertility and Sterility 2007 87, 1428-1437DOI: (10.1016/j.fertnstert.2006.11.061) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Assessment of LGR-4, LGR-5, LGR-7, and LGR-8 mRNA expression in the human myometrium using semiquantitative RT-PCR. A representative sample from the proliferative (prol) and secretory (secr) phase uterus is shown. Testis cDNA was used as positive control (+co), and myometrial RNA and water was used as negative control (−co). Both reactions were negative, and the −RT reaction is shown as an example. Krusche. LGR-4, -5, and -7 mRNA in human endometrium. Fertil Steril 2007. Fertility and Sterility 2007 87, 1428-1437DOI: (10.1016/j.fertnstert.2006.11.061) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 5 Relaxin receptor (LGR-7) protein expression in the human cyclic endometrium. A: Day 4; B: day 10, glands of the functional layer; C: day 12, glands of the functional layer; D: day 12, surface epithelium. The insert shows a representative negative control. E: Day 26, basal endometrial layer; F day 26, surface epithelium. Interference contrast microscopy; magnification × 400. Krusche. LGR-4, -5, and -7 mRNA in human endometrium. Fertil Steril 2007. Fertility and Sterility 2007 87, 1428-1437DOI: (10.1016/j.fertnstert.2006.11.061) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions