Altered placental development and intrauterine growth restriction in IGF binding protein-1 transgenic mice

Abstract

IGF binding protein-1 (IGFBP-1) is a secretory product of decidualized endometrium and a major constituent of amniotic fluid. It is thought to modulate the actions of the IGFs on trophoblast cells and is therefore potentially important in regulating placental development and fetal growth. To investigate this hypothesis, we have studied the effects of decidual IGFBP-1 excess on fetoplacental growth in transgenic mice overexpressing human IGFBP-1. Endogenous fetal IGFBP-1 overexpression is associated with a transient impairment of fetal growth in midgestation. Maternal decidual IGFBP-1 excess is also associated with impaired fetal growth in midgestation independent of fetal genotype, indicating placental insufficiency. Our data also demonstrate that amniotic fluid IGFBP-1 is derived almost exclusively from maternal sources. Decidual IGFBP-1 overexpression has a marked effect on placental development. Placental morphology is abnormal in transgenic females due to altered trophoblast invasion and differentiation. These changes result in an increase in placental mass throughout pregnancy. This study provides the first compelling in vivo evidence that IGFBP-1 plays a role in placentation and suggests that IGFBP-1 has a pathological role in preeclampsia, a disorder characterized by shallow uterine invasion and altered placental development.

Figure 1

hIGFBP-1 gene expression in maternal decidua and fetal liver. (a) hIGFBP-1 mRNA in decidua of transgenic dams was detected by RT-PCR using a primer pair that specifically amplifies a 372-bp product from hIGFBP-1 cDNA. Lane 1, no template (negative control); lane 2, transgenic mouse liver RNA (positive control); lane 3, wild-type mouse decidua RNA (negative control); lanes 4–6, total RNA extracted decidua of transgenic females sacrificed at the indicated timepoints. M, 100-bp ladder. (b) hIGFBP-1 expression in fetal liver was detected as described in a. Lane 1, no template (negative control); lane 2, transgenic adult mouse liver RNA (positive control); lane 3, wild-type adult mouse liver RNA (negative control); lane 4, wild-type fetal liver RNA; lanes 5 and 6, liver RNA from transgenic fetuses at e14.5 and e17.5, respectively. M, molecular size markers.

Figure 2

Amniotic fluid hIGFBP-1 levels across gestation. Total hIGFBP-1 levels in amniotic fluid were measured by a species-specific immunoradiometric assay. The data compare hIGFBP-1 levels between (a) wild-type and transgenic females and (b) wild-type and transgenic fetuses from F⁺/M^– matings. Data represent mean hIGFBP-1 levels in amniotic fluid from 15–25 fetuses. Black bars, transgenic; white bars, wild-type. *P <0.01 for e17.5 transgenic versus wild-type.

Figure 3

Western ligand and immunoblot analysis of amniotic fluid hIGFBP-1. (a) Western ligand blot of amniotic fluid from transgenic (tg) and wild-type (wt) fetuses from F⁺/M^– matings. Human IGFBP-1 migrates with an apparent molecular weight of 30–32 kDa. A high-molecular-weight IGFBP (40–46 kDa) whose size is consistent with that of variantly glycosylated IGFBP-3 was also detected. (b) Western immunoblot analysis of amniotic fluid from the membrane shown in a using a mouse anti–hIGFBP-1 monoclonal antibody (mAb 6305). e, embryonic day.

Figure 4

Phosphorylation profile of amniotic fluid hIGFBP-1. Amniotic fluid from F⁺/M^– matings was assayed for total and nonphosphorylated hIGFBP-1, and the phosphorylation status of the protein was expressed as the ratio of the two values (nonphosphorylated/total hIGFBP-1). Data represent the mean of 15–25 samples of amniotic fluid. *P < 0.01 for e14.5 versus e17.5.

Figure 5

Northern blot analysis of placental IGF-II expression. Total RNA from e11.5 and e14.5 placentas from wild-type (F^–/M⁺) and transgenic (F⁺/M^–) females was probed with the murine IGF-II full-length cDNA. The membranes were stripped and reprobed with murine GAPDH cDNA to verify equal sample loading.

Figure 6

Placental histology in hIGFBP-1 transgenic mice. (a) Gross morphology. The different functional layers of the mouse placenta are shown (d, decidual; j, junctional; l, labyrinthine). Differences in placental cross-sectional area and the relative proportions of the different functional layers of the placenta are clearly evident. (b) Decidual thickness. The thickness of the decidual layer is greater in placentas from wild-type females, indicating reduced trophoblast invasion in transgenic females. The labyrinthine zone is grossly enlarged, occupying approximately 80% of the cross-sectional area of the placenta compared with about 50% in wild-type placentas. sp, spongiotrophoblast; mv, maternal blood vessel; gc, glycogen cells; gt, trophoblast giant cells. (c) Trophoblasts in the junctional zone. The junctional zone is larger in placentas from transgenic females. There are similar numbers of glycogen cells and spongiotrophoblasts in placentas from wild-type females. In transgenic mice, spongiotrophoblasts are the predominant cell type; glycogen cells are relatively scarce compared with placentas from wild-type females.