Anat Cell Biol.  2024 Sep;57(3):400-407. 10.5115/acb.23.313.

Alcohol intake during pregnancy reduces offspring bone epiphyseal growth plate chondrocyte proliferation through transforming growth factor β-1 inhibition in the Sprague Dawley rat humerus

• Pillay D
• Perry V
• Ndou R

• KMID: 2559758
• DOI: http://doi.org/10.5115/acb.23.313

Abstract

Intrauterine alcohol exposure delays bone maturation and intensifies osteoporosis and fracture risk. As most studies emphasize the neurological aspects of intrauterine alcohol exposure, there is a lack of research on the implications pertaining to osseous tissue. Previous studies investigated these effects in fetuses, with limited studies on postnatal life. Postnatal studies are crucial since peak bone growth occurs during adolescence. This study aimed at assessing the effects of prenatal alcohol exposure on the humerus proximal and distal growth plate chondrocytes in 3-week-old rats. Sprague Dawley rats (n=9) were assigned to either the ethanol group (n=3), saline (n=3), and untreated (n=3) group and time-mated. Once pregnant, as confirmed by the presence of a copulation plug, the former 2 groups were treated with 0.015 ml/g of 25.2% ethanol and 0.9% saline. The untreated group received no treatment. The left humeri belonging to 6 pups per group were used. Serial sections were cut with a microtome at 5 µm thickness. These sections were stained with haematoxylin and eosin for assessment of normal morphology or immunolabeled with anti-Ki-67 and transforming growth factor β-1 (TGFβ-1) antibody. Prenatal alcohol exposure adversely effected the growth plate sizes and the number of cells in the proliferative zone. Fewer TGFβ-1 immunopositive and proliferative chondrocytes were found using the anti-Ki-67 antibody. This may explain the growth retardation in offspring exposed to gestational alcohol, showing that gestational alcohol exposure inhibits cell proliferation, aiding the diminished stature.

Keyword

Body height; Fetus; Fetal alcohol syndrome; Cell proliferation; Chondrocytes

Figure

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  Fig. 1 Study design. A diagrammatic illustration of the group allocation of dams, pups and humeri for the study.

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  Fig. 2 Photomicrographs of haematoxylin and eosin-stained sections of the epiphyseal growth plate of the humerus. (A–C) are representative micrographs of the proximal while (D–F) show the distal epiphyseal growth plate for the untreated, saline and ethanol groups, respectively. The insert is blown up from the boxed area in (A) to show proliferative zone chondrocytes. The white arrowheads point to the typical stacking of chondrocytes in the proliferative zone and point to examples of this chondrocyte stacking. Scale bar=40 microns and applies to all images.

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  Fig. 3 Epiphyseal growth place area and chondrocyte parameters in the proliferative zone illustrated as a percentage difference relative to the untreated control group. (A) Histomorphometry of the epiphyseal growth plate surface area. (B) Epiphyseal growth plate proliferative zone number of chondrocytes. (C) Ki-67 immuno-positive (proliferative) chondrocytes are shown for the proliferative zone. (D) Transforming growth factor β-1 immuno-positive chondrocytes in the epiphyseal growth plate proliferative zone. The mean percentage difference in relation to the untreated control group is shown for the saline controls and the ethanol group in the proximal and distal epiphyseal growth plate. The P-values are for comparison with the untreated control group. Error bars represent standard error of the mean.

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  Fig. 4 Photomicrograph of humerus proliferative zone chondrocytes immunolabeled with the anti-Ki-67 antibody. Representative micrographs of the proximal (A–C) and distal (D–F) epiphyseal growth plate for the three groups studied. The arrowheads point to examples of the typical stacking of chondrocytes in the proliferative zone. Scale bar=30 microns and applies to all images.

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  Fig. 5 Photomicrograph of humerus proliferative zone chondrocytes immunolabeled with the anti-transforming growth factor β-1 antibody. Representative micrographs of the proximal (A–C) and distal (D–F) epiphyseal growth plate for the three groups studied. The arrowheads point to examples of the typical stacking of chondrocytes in the proliferative zone. Scale bar=40 microns and applies to all images.

Reference

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