Expression and protein localisation of IGF2 in the marsupial placenta

Abstract

Background: In eutherian mammals, genomic imprinting is critical for normal placentation and embryo survival. Insulin-like growth factor 2 (IGF2) is imprinted in the placenta of both eutherians and marsupials, but its function, or that of any imprinted gene, has not been investigated in any marsupial. This study examines the role of IGF2 in the yolk sac placenta of the tammar wallaby, Macropus eugenii.

Results: IGF2 mRNA and protein were produced in the marsupial placenta. Both IGF2 receptors were present in the placenta, and presumably mediate IGF2 mitogenic actions. IGF2 mRNA levels were highest in the vascular region of the yolk sac placenta. IGF2 increased vascular endothelial growth factor expression in placental explant cultures, suggesting that IGF2 promotes vascularisation of the yolk sac.

Conclusion: This is the first demonstration of a physiological role for any imprinted gene in marsupial placentation. The conserved imprinting of IGF2 in this marsupial and in all eutherian species so far investigated, but not in monotremes, suggests that imprinting of this gene may have originated in the placenta of the therian ancestor.

Figure 1

IGF2 protein in the bilaminar (A and B) and trilaminar (D and E) yolk sac at day 25–26 of gestation. IgG negative controls for the bilaminar (C) and trilaminar (F) yolk sac. Staining was strongest in the trophoblast (Tr), but some endodermal (En) cells of the yolk sac placenta also stained. Staining was generally stronger in the trilaminar yolk sac and in both portions of the yolk sac staining increased later in gestation (see Fig. 2). Strong staining can be seen in the uterine epithelium (Ep) immediately adjacent to the bilaminar (avascular) yolk sac placenta. There was little staining in the mesenchyme (Me) and endothelium of large vitelline vessels (Vv) of the trilaminar (vascular) yolk sac placenta. Some stromal (St) and endothelial cells (Ed) in the maternal endometrium also stained (G and IgG negative H), as did the uterine epithelium (Ep) and some endometrial glands (Gl). Scale bar is shown at the bottom left of each image.

Figure 2

IGF2R (A & C) and IGF1R (E & G) protein in the bilaminar (BYS; A & E) and trilaminar (TYS; C & G) yolk sac at day 25. Appropriate IgG antibody negative controls for IGF2R and IGF1R antibodies are shown (B, D, F, & H). IGF2R staining was strongest in the trophoblast (Tr), with lighter staining in the yolk sac endoderm (En) and little or no staining in the mesenchyme (Me) surrounding vitelline vessels (Vv). IGF2R staining was localised in the cytoplasm and cell membrane. IGF1R stained all yolk sac cell types. Both antibodies also stained the uterine epithelium and some stromal cells in the endometrium (Endo). Scale bar is shown at the bottom left of each image.

Figure 3

Intensity of staining to IGF2 and IGF2R antibodies in the yolk sac trophoblast during the final third of gestation (A). The intensity of staining was measured subjectively as described in the experimental procedures. The bilaminar (BYS) (shaded bars) and trilaminar yolk sac (TYS) (open bars) of matched samples were assessed independently. Samples were grouped into days 19–21 (n = 4), 22–24 (n = 5), and 25–26 (n = 4). Staining intensity to the IGF2 antibody was consistently stronger in the TYS, especially at days 25–26. Staining by the IGF2 antibody was notably lighter at days 19–21 than later stages (days 22 to 26). Staining by the IGF2R antibody did not differ notably between the bilaminar and trilaminar yolk sac, nor were there marked differences corresponding to developmental stage. Intensity of staining by IGF2 and IGF2R antibodies in yolk sac cells (B). Staining intensity was noticeably higher in the trophoblast (Tr) (stippled bars) than in the yolk sac endoderm (En) (striped bars) of the bilaminar (BYS) and trilaminar (TYS) for IGF2, but not IGF2R. The staining intensity represents the average for fetal stages between days 19 and 26 (n = 13). Light background staining with the IgG antibody negative control in the yolk sac endoderm was taken into account when judging the staining intensity of the yolk sac endoderm for IGF2 and IGF2R antibodies.

Figure 4

(A) IGF2 Western blot. A single band was detected at approximately 23 kD, consistent with predicted the protein size. Non-quantitative (B) and quantitative (C) IGF2 and IGF2R RT-PCR. Tissues include adult liver, endometrium (endo), pouch young tail (PY), fetal body (fetus), bilaminar yolk sac (BYS,), trilaminar yolk sac (TYS) and allantois (all), a "no template" control (NTC) is also shown. Only the BYS and TYS were examined quantitatively and stages examined were grouped; 19–21 (n = 8), 22–24 (n = 7), and 25–26 (n = 6). IGF2 mRNA was expressed on both TYS (stippled squares), and BYS (open diamonds) but was higher in the TYS at all stages. IGF2 expression increased between days 19–21 and 22–24. IGF2R mRNA levels fluctuated, but the BYS and TYS were not significantly different. Significant differences are shown by superscript letters. Means sharing the same letters are not significantly different (P > 0.05). Means with different superscript letters are significantly different (P ≤ 0.05).

Figure 5

VEGF mRNA (open squares) relative to β-actin levels in trilaminar yolk sac during the final third of gestation (A) or in vitro (B). VEGF expression in the trilaminar yolk sac was examined at stages 19–21 (n = 8), 22–24 (n = 7), and 25–26 (n = 6). A gradual increase in VEGF expression is evident and by term (days 25–26) expression was significantly higher than days 19–21 (t-test, one-way, equal variance, P= 0.002, F-test = 0.165). Trilaminar yolk sac explants were cultured for 8 (n = 4) and 18 (n = 5) hours with hr-IGF2 (treatment: stippled bars) or in media only (control: open bars). VEGF expression was consistently higher in IGF2 treated explants (see text).