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3.3.2.1 Endocrine IGF system

Endocrine levels of the IGF system were determined in fetal and maternal blood to determine if similar changes in our mouse undernutrition model are similar to changes to endocrine levels of the IGF system documented in human pregnancies afflicited with fetal growth restriction. In maternal plasma of 70% MNR, IGF-I and IGFBP-3 concentrations were significantly decreased (Fig. 3.1; IGF-I: control 56 ± 12 ng/mL vs. MNR 26 ± 4 ng/mL; P < 0.05; IGFBP-3: control 1048 ± 70 ng/mL vs. MNR 586 ± 84

TABLE 3.4 Effect of Maternal Nutrient Restriction beginning at E 6.5 of pregnancy on placental and fetal tissue weights at E 18.5.

Control (n=24) 80% MNR (n=8) 70% MNR (n=9) 60% MNR (n=7)

Fetus 1397 ± 17 1149 ± 64a 894 ± 52a,b 967 ± 63a,b Placenta 95 ± 3 91 ± 5 72 ± 3a,b 78 ± 4a Heart 8 ± 1 6 ± 1a 5 ± 1a 5 ± 1a Lung 51 ± 1 39 ± 3a 29 ± 2a,b 32 ± 2a Liver 91 ± 2 69 ± 5a 46 ± 3a,b 50 ± 3a,b Gut 81 ± 1 67 ± 4a 48 ± 4a,b 55 ± 5a Kidney 12 ± 1 9 ± 1a 7 ± 1a 8 ± 1a Brain 69 ± 1 64 ± 3 56 ± 2a 58 ± 3a Carcass 977 ± 12 801 ± 4a 644 ± 38a,b 673 ± 50a Litter Size 13 ± 1 11 ± 1 11 ± 1 10 ± 1a Pregnant mothers were fed ad libitum (control) or 80%, 70% or 60% MNR beginning at E 6.5. At E 18.5, mothers were sacrificed and placenta and fetal tissues were weighed for each fetus for a mean litter value. The result, litter mean ±SEM for ‘N’ number of litters, is expressed in mg.

Superscripts (a = different from control ( P < 0.05); b = different from 80% MNR ( P < 0.05); c = different from 70% MNR ( P < 0.05) among treatment groups indicate a significant difference in weight by one-way ANOVA (P < 0.05), followed by Tukey’s multiple comparisons test.

Figure 3.1 The effect of maternal nutrient restriction on circulating concentrations

of the IGF system in the maternal and fetal plasma at E18.5. (A) Maternal plasma

IGF-I concentrations (mean ± SEM) were 56 ± 12 ng/mL in controls (N=8) and 26 ± 4 ng/mL in 70% MNR (N=8) (P<0.05). IGF-I concentrations were 55% lower in 70% MNR mothers (P < 0.05). (B) Maternal plasma concentrations (mean ± SEM) of IGFBP- 1, -2 and -3 in controls (N=8) and 70% MNR (N=8). In 70% MNR mothers, the IGFBP-1 concentration of 899 ± 201 ng/mL (vs 68 ± 17 ng/mL in controls) and IGFBP-2 concentration of 3,299 ± 432 ng/mL (vs 1648 ± 253 ng/mL in controls) were significantly higher (both P < 0.005), while IGFBP-3 concentration of 586 ± 84 ng/mL (vs 1048 ± 70 ng/mL in controls) was significantly lower (P < 0.001). (C) Fetal plasma IGF-I concentrations (mean ± SEM) were 29 ± 3 ng/mL in controls (N=8) and 20 ± 2 ng/mL in 70% MNR (N=8). IGF-I concentrations were 30% lower in 70% MNR fetuses (P < 0.05). (D) Fetal plasma concentrations (mean ± SEM) of IGFBP-1, -2 and -3 in controls (N=8) and 70% MNR (N=8). In 70% MNR fetuses, the IGFBP-1 concentration of 362 ± 70 ng/mL (vs 197 ± 25 ng/mL in controls) and IGFBP-2 concentration of 2719 ± 170 ng/mL (vs 2194 ± 169 ng/mL in controls) were significantly higher (both P < 0.05), while IGFBP-3 concentration of 1475 ± 112 ng/mL (vs 1944 ± 111 ng/mL in controls) was significantly lower (P < 0.01).

ng/mL; P < 0.001), whereas IGFBP-1 and IGFBP-2 concentrations were significantly increased (Fig. 3.1; IGFBP-1: control 68 ± 17 ng/mL vs. MNR 899 ± 201 ng/mL; P < 0.005; IGFBP-2: control 1648 ± 253 ng/mL vs. MNR 3299 ± 432 ng/mL; P < 0.005). In fetal plasma, changes to concentrations of the IGF system peptides were similar to that of maternal plasma with lower IGF-I and IGFBP-3 concentrations and higher IGFBP-1 and IGFBP-2 concentrations in MNR fetuses compared to controls (Fig. 3.1; IGF-I: control 29 ± 3 ng/mL vs. MNR 20 ± 2 ng/mL; P < 0.05; IGFBP-3: control 1944 ± 111 ng/mL vs. MNR 1475 ± 112 ng/mL; P < 0.01; IGFBP-1: control 197 ± 25 ng/mL vs. MNR 362 ± 70 ng/mL; P < 0.05; IGFBP-2: control 2194 ± 169 ng/mL vs. MNR 2719 ± 170 ng/mL; P < 0.05). The ratio of IGF-I to IGFBP-3 was similar between control and 70% MNR in both fetal and maternal plasma. Assays for IGF-II levels in plasma are unavailable for reliable estimates in the small volumes of mouse plasma.

3.3.2.2 IGF system mRNAs in fetal tissues and placentae

The expression levels of mRNAs encoding the IGF system in different fetal organs/tissues were performed to determine autocrine/paracrine changes of the system that correspond with changes in organ/tissue growth, and the organs/tissues that are responsible for the changes in fetal endocrine levels of the IGF system. IGF-I, -II, and IGFBP-1,-2,-3 mRNA levels were determined in heart, lung, liver, brain, carcass and placenta in control and 70% MNR litters using qRT-PCR. The liver demonstrated greatest response to MNR with significantly increased levels of IGFBP-1, IGFBP-2, and IGFBP-3 mRNAs (TABLE 3.5). Placenta, lung and brain demonstrated an increase in IGFBP-3 mRNA levels (TABLE 3.5). The placenta was the only organ that showed a

TABLE 3.5 Effect of 70% Maternal Nutrient Restriction beginning at E 6.5 of pregnancy on Insulin-Like Growth Factor (IGF) System mRNA levels at E 18.5 in placental and fetal tissues.

IGF-I IGF-II IGFBP-1 IGFBP-2 IGFBP-3

Placenta 0.53 ± 0.12* 1.11 ± 0.07 0.64 ± 0.20 1.10 ± 0.16 1.76 ± 0.12* Heart 0.58 ± 0.08 1.04 ± 0.09 - - 1.24 ± 0.13 Lung 0.71 ± 0.11 0.93 ± 0.09 1.85 ± 0.80 0.97 ± 0.21 2.22 ± 0.51* Liver 0.70 ± 0.10 1.04 ± 0.11 5.10 ± 1.86* 1.86 ± 0.33* 1.29 ± 0.08* Brain 0.80 ± 0.09 0.88 ± 0.09 2.29 ± 0.50 0.90 ± 0.08 1.48 ± 0.13* Carcass 0.76 ± 0.06 1.14 ± 0.10 - 1.60 ± 0.20 1.45 ± 0.15

Pregnant mothers were fed ad libitum (control) or 70% MNR beginning at E 6.5. At E 18.5 mothers were sacrificed and placenta and fetal tissues collected and pooled by litter. The IGF system mRNA levels were measured by quantitative real-time PCR and corrected for the internal housekeeping gene, β2 macroglobulin. The relative mRNA levels of each tissue of 70% MNR group (N = 6 litters) are expressed relative to those of control group (N = 6 litters), which are assigned a value of one.

* Indicates a significant fold-change between control and 70% MNR groups by Mann- Whitney Test (P < 0.05).

significant reduction in IGF-I mRNA level. In most fetal tissues, IGF-I mRNA levels were relatively lower, with no change in IGF-II mRNA levels (TABLE 3.5). At E11.5, the liver is the major source of hematopoietic cells during gestation as it is populated by hematopoietic stem cells and provides a developmental niche for the expansion, maturation and enucleation of primitive erythroid cells (22). Late in development, from approximately E16 onwards, the site of hematopoiesis shifts gradullay to the bone marrow, which is the definitive site where hematopoietic stem cells and progenitors of the adult hematopoietic system reside (22). In situ hybridization demonstrated IGF-I and - II and IGFBP-1, -2 and -3 mRNAs to be expressed in hepatocytes and not in hematopoietic cells (Fig. 3.2 and Fig. 3.3) (Appendix Fig. 1.1, Fig 1.2 and Fig. 1.3).