Placental insufficiency and heavier placentas in sheep after suppressing CXCL12/CXCR4 signaling during implantation†

Abstract

During implantation, trophoblast cell invasion and differentiation is predominantly important to achieving proper placental formation and embryonic development. The chemokine, C-X-C motif chemokine ligand 12 (CXCL12) working through its receptor C-X-C motif chemokine receptor 4 (CXCR4) is implicated in implantation and placentation but precise roles of this axis are unclear. Suppressing CXCL12/CXCR4 signaling at the fetal-maternal interface in sheep reduces trophoblast invasion, disrupts uterine remodeling, and diminishes placental vascularization. We hypothesize these negative impacts during implantation will manifest as compromised fetal and placental growth at midgestation. To test, on day 12 postbreeding, osmotic pumps were surgically installed in 30 ewes and delivered intrauterine CXCR4 inhibitor or saline for 7 or 14 days. On day 90, fetal/maternal tissues were collected, measured, weighed, and maternal (caruncle) and fetal (cotyledon) placenta components separated and analyzed. The objectives were to determine if (i) suppressing CXCL12/CXCR4 during implantation results in reduced fetal and placental growth and development and (ii) if varying the amount of time CXCL12/CXCR4 is suppressed impacts fetal/placental development. Fetal weights were similar; however greater placental weight and placentome numbers occurred when CXCL12/CXCR4 was suppressed for 14 days. In caruncles, greater abundance of fibroblast growth factor 2, vascular endothelial growth factor A, vascular endothelial growth factor A receptor 1 (FLT-1), and placental growth factor were observed after suppressing CXCL12/CXCR4. Similar results occurred in cotyledons except less vascular endothelial growth factor in 7 day group and less fibroblast growth factor in 14 day group. Our data underscore the importance of CXCL12/CXCR4 signaling during placentation and provide strong evidence that altering CXCL12-mediated signaling induces enduring placental effects manifesting later in gestation.

Keywords: CXCL12; CXCR4; PLGF; VEGF; caruncle; cotyledon; placental insufficiency; preeclampsia; sheep; trophoblast.

Graphical Abstract

Figure 1

Suppressing CXCL12/CXCR4 for 7 or 14 days during implantation did not impact fetal weights on day 90 of gestation. Fetal weights were recorded on day 90 of gestation after ewes received intrauterine PBS (CON) or CXCR4 inhibitor, AMD3100 (AMD) for 7 days (A) or 14 days (B) during implantation. Data are represented by the mean fetal weight ± SEM.

Figure 2

Greater number of placentomes and placental weight on day 90 of pregnancy after suppressing CXCL12/CXCR4 axis at fetal-maternal interface for 14 days during implantation. Placentome numbers, weights, and morphological type were recorded on day 90 of gestation after ewes received intrauterine PBS (CON) or CXCR4 inhibitor, AMD3100 (AMD) for 7 days (A–C) or 14 days (D–F) during implantation. Data are represented by the mean placentome weight or placentome numbers and classification ± SEM, and significance denoted with an asterisk (^**) when P < 0.01.

Figure 3

Suppressing CXCL12/CXCR4 for 14 days during implantation results in placental insufficiency on day 90 of gestation. Placental efficiency calculated as the ratio of fetal weight (g) per placental weight (g) on day 90 of gestation for ewes that received intrauterine PBS (CON) or CXCR4 inhibitor, AMD3100 (AMD) for 7 days (A) or 14 days (B) during implantation. Data are represented by the mean fetal/placental weight ratio ± SEM, and significance denoted with an asterisk (^**) when P < 0.01.

Figure 4

Disrupting CXCL12/CXCR4 signaling at fetal maternal interface during implantation for 7 or 14 days results in altered abundance of growth factors and receptors in maternal (caruncle) and fetal (cotyledon) placenta on day 90 of gestation. Immunoblotting used to detect protein abundance for select angiogenic/growth factors and receptors in caruncles and cotyledons from ewes that received intrauterine PBS (CON) or CXCR4 inhibitor, AMD3100 (AMD) for 7 days (A, C) or 14 days (B, D) during implantation. Data represented by the mean ± SEM and each graph is followed by a representative immunoblot. Significance denoted with an asterisk when P < 0.05 (^*), P < 0.01 (^**), P < 0.001 (^***), and tendency acceptance at P < 0.1 (#).

Figure 5

Suppressing CXCL12/CXCR4 signaling at fetal maternal interface for 7 days during implantation results in greater immunolocalization of vWF and increased BV circumference in placentomes on day 90 of pregnancy. A representative cartoon schematic of the placentome separated into three levels is depicted in panel (A). Greater immunoreactive vWF protein was observed in all placentome levels from ewes treated with CXCR4 inhibitor, AMD3100 (AMD) for 7 days (B) compared with PBS (CON). Number of BV was similar in placentomes (D) whereas BV circumference increased in L1, and the whole placentome on average (F). Arrows point to BVs in placentomes from representative 20× magnification (100 μm) images for CON (C) and AMD (E). Red fluorescence signifies vWF localization and blue represents 4,6-diamidino-2-phenylindole (DAPI) counterstaining of nuclei. Significance denoted with an asterisk when P < 0.05 (^*), P < 0.01 (^**), and tendency acceptance at P < 0.1 (#).

Figure 6

Suppressing CXCL12/CXCR4 signaling at fetal maternal interface for 14 days during implantation results in greater vWF placentome immunolocalization, BVs numbers, and larger vessel circumference in placentomes on day 90 of pregnancy. A representative cartoon schematic of the placentome separated into three levels is depicted in panel (A). Greater immunoreactive vWF protein was observed in all placentome levels from ewes treated with CXCR4 inhibitor, AMD3100 (AMD) for 14 days (B) compared with PBS (CON). Number of BVs increased in L1, L2, and the whole placentome on average (D). BV circumference increased in L1, L3, and the whole placentome on average (F). Arrows point to BV in placentomes from representative 20× magnification (100 μm) images for CON (C) and AMD (E). Red fluorescence signifies vWF localization and blue represents 4,6-diamidino-2-phenylindole (DAPI) counterstaining of nuclei. Significance denoted with an asterisk when P < 0.05 (^*).