Methyl donor deficiency affects fetal programming of gastric ghrelin cell organization and function in the rat

Abstract

Methyl donor deficiency (MDD) during pregnancy influences intrauterine development. Ghrelin is expressed in the stomach of fetuses and influences fetal growth, but MDD influence on gastric ghrelin is unknown. We examined the gastric ghrelin system in MDD-induced intrauterine growth retardation. By using specific markers and approaches (such as periodic acid-Schiff, bromodeoxyuridine, homocysteine, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling, immunostaining, reverse transcription-polymerase chain reaction), we studied the gastric oxyntic mucosa cellular organization and ghrelin gene expression in the mucosa in 20-day-old fetuses and weanling pups, and plasma ghrelin concentration in weanling rat pups of dams either normally fed or deprived of choline, folate, vitamin B6, and vitamin B12 during gestation and suckling periods. MDD fetuses weighed less than controls; the weight deficit reached 57% at weaning (P < 0.001). Both at the end of gestation and at weaning, they presented with an aberrant gastric oxyntic mucosa formation with loss of cell polarity, anarchic cell migration, abnormal progenitor differentiation, apoptosis, and signs of surface layer erosion. Ghrelin cells were abnormally located in the pit region of oxyntic glands. At weaning, plasma ghrelin levels were decreased (-28%; P < 0.001) despite unchanged mRNA expression in the stomach. This decrease was associated with lower body weight. Taken together, these data indicate that one mechanism through which MDD influences fetal programming is the remodeling of gastric cellular organization, leading to dysfunction of the ghrelin system and dramatic effects on growth.

Figure 1

Photographs of stomach (upper panel) and body weight, plasma vitamin B12, B9, and Hcy variations (mean ± SEM; lower panel) in control (C) and MDD pups at weaning. ***P < 0.001 between control and deficient rats.

Figure 2

Immunohistological examination of the gastric oxyntic mucosa in control (A, C, E) and MDD rats (B, D, F) at weaning. Mucous cells labeling by PAS staining (A, B). Apoptosis detection by TUNEL assay: in control, pit cells, some neck cells (black arrowheads) and cells from the base region were TUNEL-positive (C), whereas in MDD apoptotic cells were found throughout the mucosa (D). BrdU labeling of proliferative cells: In control rats, newly proliferating cells were located in the isthmus region (E, white arrowheads) and migrated to the base, whereas in MDD pups, BrdU-positive cells were mainly located from the neck to the pit region (F, white arrows); thickness of the total gastric oxyntic mucosa and of the surface mucin staining (G); quantification of proliferative (BrdU positive) and apoptotic (TUNEL) cells in the gastric oxyntic mucosa (H). MM, muscularis mucosa; LP, lamina propria; GL, gastric lumen. **P < 0.01.

Figure 3

Immunohistological localization of specific cell type among the gastric glands in control and MDD E20 fetuses (four left panels) and rats at weaning (four right panels). A–D: Mucus-secreting pit cells labeled with UEA1 lectin are colored in red, whereas mucus-secreting neck cells labeled with GSII lectin are colored in green. E–H: Parietal cells are colored in green. LP, lamina propria; GL, gastric lumen.

Figure 4

Localization of enteroendocrine ghrelin-producing cells colored in red (A–F, white arrows and arrowheads) and of zymogenic IF secretory cells (G–J; red) in control and MDD E20 fetuses (four left panels) and rats at weaning (six right panels). An enlarged view of the areas in squares (F and J) is shown in a supplementary figure (see Supplemental Figure S1 at http://ajp.amjpathol.org). LP, lamina propria; GL, gastric lumen.

Figure 5

Plasma concentration of ghrelin (mean ± SEM in pg/ml) and gastric ghrelin expression in control and MDD pups at weaning. ***P < 0.001 between control and deficient rats.