The human relaxin receptor (LGR7): expression in the fetal membranes and placenta

Abstract

The relaxin receptor has been recently described as a leucine-rich repeat G-protein coupled receptor and designated as LGR7. A closely related receptor, LGR8, is co-expressed by some cells. This study explored the expression of the genes for these receptors in the human fetal membranes and placenta by RT-PCR and the LGR7 protein by immunolocalization. The results showed that LGR7 was well expressed in the fetal membranes, with significantly more in the decidua (p<0.05) than in the amnion. On the other hand, relatively low levels were expressed in the placenta. The major splice variant of LGR7 was undetectable in either the placenta or fetal membranes. Expression of LGR8 was also below the level of detectability in either tissue. Immunostaining for LGR7 was conducted with antisera to both its endodomain and ectodomain, in order to seek evidence for a solubilized ectodomain. However, similar staining patterns were obtained with both antisera, with predominant staining in the cells of the amniotic epithelium, chorionic cytotrophoblast and decidua. Full-thickness fetal membranes from preterm deliveries, before and after labor or after preterm premature rupture of the membrane (PPROM) and labor were collected. In addition, membranes at term, both before and after spontaneous labor were used for analysis of LGR7 gene expression. There was significantly greater LGR7 expressed (p=0.01) in the preterm period compared to term, indicating a potentially important role for relaxin at this time. There was a marginal decline in LGR7 gene expression after labor and delivery both at preterm and term, which did not reach significance. Immunostaining patterns showed less inter-patient variability than did gene expression, with more intense staining for LGR7 after labor and delivery.

Figure 1

LGR7 gene expression in the amnion, chorion, decidua and placenta collected at term (A) before labor after elective Cesarean section (n = 5) and (B) after spontaneous labor and delivery (n = 3). None of these tissues had any histological evidence of inflammation or infection. Real-time PCR data were normalized to the expression of 18S in each sample and are shown as relative gene expression ± SEM compared to the expression in the placentas of each group. (A) *Significantly (p < 0.05) more LGR7 expressed in the decidua before labor compared to the amnion or placenta and (B) *significantly (p < 0.05) more in the decidua after labor compared to the amnion. There were relatively low levels of LGR7 expressed in the placenta both before and after labor.

Figure 2

PCR products are shown on a gel when amplified with different primer sets for LGR7 (A) and LGR8 (B). Samples of RNA from three placentas and fetal membranes (different patients) were amplified with primers designed for LGR7 real-time PCR (A, lanes 1–3, placentas; lanes 4–6, membranes), a single band of 100 bp was detected in both tissues. Primers designed to detect both LGR7 and its splice variant (A, lanes 8–10, placenta; lanes 11–13 membranes) show that only LGR7 was detected at 200 bp. Lane 7 shows molecular size markers. The same RNA samples were also amplified with primers specific for LGR8 (B, lanes 1–3, placentas; lanes 4–6, membranes), its transcript was undetectable in RNAs from either tissue, while RNA from human uterus (lane 7), shows a single band at 100 bp corresponding to LGR8. Molecular size markers are shown in lane 8.

Figure 3

Immunostaining for LGR7 in a representative fetal membrane of a patient after spontaneous labor and delivery at term. Consecutive sections were stained with an antiserum to the endodomain (A, C, E, G) and the ectodomain (B, D, F, H) of LGR7. The amniotic epithelium (A, B) and mesenchyme of the amniotic connective tissue (C, D) showing more staining in the mesenchyme with the antiserum to the ectodomain (D) compared to the endodomain (C). The cells of the chorionic cytotrophoblast show strong cytoplasmic staining (E and F), as do those of the parietal decidua (G and H). Magnification: ×600.

Figure 4

LGR7 gene expression by real-time PCR in full-thickness fetal membranes collected at preterm Cesarean section and no labor, after labor and delivery and from patients with PPROM (n = 4 of each) and at term, before and after spontaneous labor (n = 3 of each). Data are presented as the n-fold difference relative to term labor, LGR7 expression ± SEM. A 5-fold decline in expression was seen after labor at term and expression was halved after preterm labor. These were not significant due to the large inter-patient variability. However, tissues from PPROM patients remained elevated although they had experienced labor and delivery. * Disregarding the mode of delivery, there was significantly more LGR7 expressed (p = 0.01) at preterm than at term.

Figure 5

Immunostaining for LGR7 in a representative fetal membrane from a patient before labor at term, stained with an antiserum to the (A) endodomain (1:800) and (B) ectodomain (IgG: 6 μg/ml) of LGR7. In a patient after normal spontaneous labor and delivery at term stained with an antiserum to the (C) endodomain and (D) ectodomain. Controls are membranes from the patient before labor stained with (E) non-immune serum and (F) non-immune IgG, at the same concentrations as the primary antiserum/IgG. Staining patterns were similar with both antisera, but show more intense staining after (C and D) compared to before labor and delivery (A and B). Identification of the major cell layers is shown in (F); (a) = amniotic epithelium, (ch) = chorionic cytotrophoblast and (d) = decidua. Magnification: ×200.

Figure 6

LGR7 gene expression by real-time PCR in full-thickness fetal membrane explants (n = 4 patients) treated with human relaxin for 6 h. Data presented relative to expression in the control ± SEM. A dose–response increase in the expression of LGR7 up to 25 ng/ml relaxin was evident but failed to reach significance.