Maternal protein restriction regulates IGF2 system in placental labyrinth

Abstract

This study was to test the hypothesis that altered IGF2 system in the placental labyrinth zone (LZ) impairs feto-placental growth in response to maternal protein restriction. Rats were fed a 20% protein diet and an isocaloric 6 % protein diet (LP) from day 1 to days 14, 18, or 21 of pregnancy. The effects of diet, gender of placenta and fetus, and day of pregnancy on placental weight, fetal weight, and expression of the IGF2 axis in the placental LZ and amino acids in maternal plasma were analyzed. Growth restriction occurred in both female and male fetuses by LP, coincident with impaired LZ growth and efficiency. The expression of Igf2, Igf2P0, Igf1r, Igf2r, Insr, Igfbp1, and Igfbp2 in placental LZ were affected by diet, gender and/or day of pregnancy. Concentrations of total essential amino acids and total nonessential amino acids were reduced and increased, respectively, in maternal plasma of LP-fed rats. These results indicate that adaptation of the IGF2 system in rat LZ occurs in a sex- and time-dependent manner in response to maternal protein restriction; however, these adaptations cannot prevent the growth restriction of both male and female fetuses during late pregnancy.

Figure 1

Gender determination by PCR for Sry gene. Partial Sry gene with a size of 300bp was amplified in PCR with genomic DNA from male, but not female, fetal extraembryonic membrane from mothers fed a normal diet (CT) or low protein diet (LP) at day 18 of pregnancy. Genomic DNA from tails of adult males and females were included as positive and negative controls, respectively.

Figure 2

Expression of Igf2 and placental specific transcript Igf2P0 in the rat labyrinth zone (LZ). A) The expression of Igf2 was affected by day x diet (P < 0.05), with reduced expression in both the male and female LZ at days 14 and 18 of pregnancy. The expression of Igf2 was greater in the male LZ than the female LZ (P < 0.05). B) The expression of Igf2P0 was affected by day (P < 0.0001), with the greatest value at day 18 of pregnancy followed by days 21 and 14. C) The expression of Igf2P0 was greater in the male LZ compared to the female LZ (P < 0.01). D) The ratio of Igf2P0 to Igf2 transcripts in LZ was significantly lower at day 21 of pregnancy than at days 14 and 18 (P < 0.0001) and was increased by LP (P < 0.05). *P < 0.05; ***P < 0.0001

Figure 3

Western blotting on IGF2 in rat labyrinth zone. Mature IGF2 (7KD) and big IGF2 (11-17KD) were detectable in the rat LZ. The relative amount of mature IGF2 was increased at day 21 of pregnancy. The relative amount of big IGF2 was increased by LP at day 14 of pregnancy, but decreased by LP at day 21 (Day x Diet, P < 0.001). *P < 0.05; **P < 0.001

Figure 4

Expression of Igf1r, Igf2r and Insr in the rat labyrinth zone (LZ). A) The expression of Igf1r was increased with the progress of pregnancy (Day, P < 0.01) and greater in the male LZ compared to female (P < 0.05); B) The expression of Insr in LZ was decreased at day 21 of pregnancy compared to days 14 and 18 (P < 0.05) and was not altered by diet and gender of placenta. C) The expression of Igf2r was greater at days 18 and 21 (Day, P < 0.0001). D) The expression of Igf2r was greater in the male LZ (Gender, P < 0.05). E) The expression of Igf2r was reduced by LP (Diet, P < 0.05). *P < 0.05; ***P < 0.0001

Figure 5

Expression of Igfbp1 and Igfbp2 in the rat labyrinth zone (LZ). A) The expression of Igfbp1 was not affected by day of pregnancy, diet or gender due to the huge variations within the group. At day 14 of pregnancy, the expression of Igfbp1 tended to be higher compared to days 18 and 21 (P = 0.07), mainly due to the high values in both the female and male LZ treated by LP. B) The expression of Igfbp2 was decreased by LP in the male LZ at day 14 of pregnancy, not changed at day 18 of pregnancy, and increased at day 21 of pregnancy (Day x Diet, P < 0.01). At day 21 of pregnancy, LP tended to increase the expression of Igfbp2 in the female LZ (P = 0.06). *P < 0.05

Figure 6

Western blotting analysis of IGFBP1 in the rat labyrinth zone (LZ). A) Two main IGFBP1 variants in the sizes of 29KD and 55KD, respectively, were detectable by Western blotting. B) At day 14 of pregnancy, the amount of 29KD protein was increased by LP (P < 0.05). C) At day 14 of pregnancy, the amount of 55KD protein was increased 1.68-fold (P < 0.001) and 1.56-fold (P < 0.05) by LP in the female and male LZ, respectively. At day 18 of pregnancy, the amount of 55KD protein was increased 2.22-fold (P < 0.05) by LP in the female LZ. In contrast, at day 21 of pregnancy, the amount of 55KD protein was decreased 2.24-fold (P < 0.05) by LP in the male LZ. *P < 0.05; **P < 0.001.

Figure 7

Western blotting analysis of IGFBP2 in the rat labyrinth zone (LZ). A) A main IGFBP2 variant in a size of 36KD was detectable by Western blotting. B) At day 14 of pregnancy, the amount of IGFBP2 was increased 1.85-fold by LP in the female LZ (P < 0.0001) and 1.57-fold in the male LZ (P < 0.05). At day 18 of pregnancy, the amount of IGFBP2 was increased 2.72-fold (P < 0.05) by LP in the female LZ but not changed in the male LZ. At day 21 of pregnancy, the amount of IGFBP2 was decreased 1.93-fold (P < 0.05) by LP in the male LZ.