Expression and location of alpha-fetoprotein during rat colon development

Abstract

Aim: To investigate the expression of alpha-fetoprotein (AFP), a cancer-associated fetal glycoprotein, and its involvement during rat colon development.

Methods: Colons from Sprague-Dawley rat fetuses, young and adult (8 wk old) animals were used in this study. Expression levels of AFP in colons of different development stage were detected by reverse-transcriptase PCR (RT-PCR) and Western blotting. To identify the cell location of AFP in the developing rat colons, double-immunofluorescent staining was performed using antibodies to specific cell markers and AFP, respectively.

Results: The highest levels of AFP mRNA were detected in colons of rats at embryonic day 18.5 (e18.5). Compared to e18.5 d, the AFP expression was significantly decreased during rat development [85% for e20.5, P < 0.05, 58% for postnatal day 0.5 (P0.5), P < 0.05, 37% for P7, P < 0.05, 24% for P14, P < 0.05, and 11% for P21, P < 0.05] and undetected in adult rats. Only the 72-kDa isoform of AFP was detected by Western blotting, the expression pattern was similar to AFP mRNA and conformed to the results of mRNA expression. The AFP positive staining was identical to different distribution patterns in fetuses, young and adult animals and positive staining for both AFP and vimentin was overlapped in mesenchymal cells at each stage tested.

Conclusion: This study has for the first time demonstrated that AFP is localized in the mesenchyme of rat colon from the embryo to the weaning stage by immunofluorescence and presents 72-kDa isoform in the developing rat colons by Western blotting. The dynamic expression of AFP in the various developmental stages of the colon indicates that AFP might be involved in many aspects of colon development.

Figure 1

Expression of AFP mRNA using RT-PCR analysis in the developing rat colons as indicated in lanes e18.5, e20.5, P0, P7, P14, P21 and adult rats. The position of molecular weight markers is indicated on the left. A: The highest levels of AFP mRNA were detected in colons of rats at e18.5 and declined steadily during rat development and were undetected in adult rats; B: Results are indicated in percentages above the e18.5 value and are representative of three independent experiments. ^aP < 0.05 vs e18.5, e20.5 and P0, respectively.

Figure 2

Expression of AFP protein using Western blot analysis in the developing rat colons as indicated in lanes e18.5, e20.5, P0, P7, P14, P21 and adult rats. A: Western blot analysis using AFP (C-19), an affinity purified goat polyclonal antibody against a peptide mapping at the C-terminus of AFP, revealed a 72-kDa isoform. The highest levels of AFP protein were detected in colons of rats at e18.5 and declined steadily during rat development and were undetected in adult rats. B: Results are indicated in percentage above the e18.5 value and are representative of three independent experiments. ^aP < 0.05 vs e18.5, e20.5 and P0, respectively.

Figure 3

Immunofluorescence localization of AFP in the developing rat colons. A: In e18.5, AFP positive staining can be detected in the epithelium and mesenchymal tissues; B: At P0, positive cells were located at the base of the crypts and scattered on the epithelium; C, D: Only a few positive cells restricted to the base of the crypts between 14 and 21 d, and no positive cells can be detected in adult rat colons. (× 200). C, D: Only a few positive cells restricted to the base of the crypts between 14 and 21 d, and no positive cells can be detected in adult rat colons. (× 200).

Figure 4

Immunofluorescence localization of AFP and vimentin in the developing rat colons. Labeling by the AFP antibody was detected with an FITC (green)-labeled secondary antibody. Labeling of vimentin was detected with a rhodamine- (red)-labeled secondary antibody on the same section. The overlap of AFP (green) and vimentin (red) labeling appeared orange in color. Double-labeling revealed complete localization of AFP and vimentin in the same colon cells at both e20.5 and P7. A: e20.5; B: P7.