Sexually dimorphic effects of prenatal alcohol exposure on the murine skeleton

Abstract

Background: Prenatal alcohol exposure (PAE) can result in lifelong disabilities known as foetal alcohol spectrum disorder (FASD) and is associated with childhood growth deficiencies and increased bone fracture risk. However, the effects of PAE on the adult skeleton remain unclear and any potential sexual dimorphism is undetermined. Therefore, we utilised a murine model to examine sex differences with PAE on in vitro bone formation, and in the juvenile and adult skeleton.

Methods: Pregnant C57BL/6J female mice received 5% ethanol in their drinking water during gestation. Primary calvarial osteoblasts were isolated from neonatal offspring and mineralised bone nodule formation and gene expression assessed. Skeletal phenotyping of 4- and 12-week-old male and female offspring was conducted by micro-computed tomography (µCT), 3-point bending, growth plate analyses, and histology.

Results: Osteoblasts from male and female PAE mice displayed reduced bone formation, compared to control (≤ 30%). Vegfa, Vegfb, Bmp6, Tgfbr1, Flt1 and Ahsg were downregulated in PAE male osteoblasts only, whilst Ahsg was upregulated in PAE females. In 12-week-old mice, µCT analysis revealed a sex and exposure interaction across several trabecular bone parameters. PAE was detrimental to the trabecular compartment in male mice compared to control, yet PAE females were unaffected. Both male and female mice had significant reductions in cortical parameters with PAE. Whilst male mice were negatively affected along the tibial length, females were only distally affected. Posterior cortical porosity was increased in PAE females only. Mechanical testing revealed PAE males had significantly reduced bone stiffness compared to controls; maximum load and yield were reduced in both sexes. PAE had no effect on total body weight or tibial bone length in either sex. However, total growth plate width in male PAE mice compared to control was reduced, whilst female PAE mice were unaffected. 4-week-old mice did not display the altered skeletal phenotype with PAE observed in 12-week-old animals.

Conclusions: Evidence herein suggests, for the first time, that PAE exerts divergent sex effects on the skeleton, possibly influenced by underlying sex-specific transcriptional mechanisms of osteoblasts. Establishing these sex differences will support future policies and clinical management of FASD.

Keywords: Bone; Murine model; Prenatal alcohol exposure (PAE); Sexual dimorphism.

Fig. 1

Schematic of experimental design. Pregnant dams were exposed to 5% ethanol in their drinking water (prenatal alcohol exposure (PAE)) or normal drinking water. Offspring were sacrificed at postnatal (p) days 3–5 for primary osteoblast cultures, or 4- and 12-weeks of age for skeletal phenotyping. Created with Biorender.com

Fig. 2

PAE reduces in vitro bone formation and results in sex-dependent differences in osteoblast gene expression. (A) Mineralised bone nodule area of primary calvarial osteoblasts from PAE male and female mice as a percentage of the control cultures. (B) Representative whole-well scans and light microscopy images of mineralised nodules. (C) RT-qPCR of key genes identified by RT²-profiler analysis (Vegfa, Vegfb, Flt1, Tgfbr1, Bmp6, Ahsg, and Bglap) on days 7 and 14 of culture from osteoblasts derived from male control and PAE, and (D) female control and PAE mice (data normalised to β-actin). Data are presented as mean ± SEM with points showing individual experiments. *= p < 0.05, **= p < 0.01, ***= p < 0.001. Scale bars: whole well = 0.5 cm, microscopy = 200 μm

Fig. 3

PAE has a detrimental effect on the trabecular microarchitecture in male mice. µCT analysis of the tibial (A) tissue volume, (B) bone volume, (C) bone volume to tissue volume ratio (BV/TV), (D) bone surface, (E) intersection surface, (F) trabecular number (Tb.N), (G) fractal dimension, (H) connectivity density (Conn.Dn) and (I) trabecular pattern factor (Tb.Pf) in male and female PAE and control mice. (J) Representative 3D-reconstructed images of the trabecular region in control and PAE mice. Data are presented as mean ± SEM with points showing individual animals. *= p < 0.05, ***= p < 0.001, ****= p < 0.0001

Fig. 4

PAE leads to spatial variation in changes in tibial cortical parameters and geometry. Measurement and statistical analysis heat map for (A) tissue area (T. Ar), (B) tissue perimeter (T.Pm), (C) Bone area (B.Ar), (D) resistance to torsion (J), (E) maximum second moments of inertia (I_max) and (F) minimum second moments of inertia (I_min) in male and female PAE and control mice. Line graphs represent mean ± SEM for male control (black), male PAE (orange), female control (green) and female PAE (blue) mice. Graphical heat map summarises statistical differences at specific matched locations along the tibial length (10–90%), for the PAE effect in females (Ctl-PAE F) and males (Ctl-PAE M), and the sex effect in control (F-M Ctl) and PAE (F-M PAE) groups. Red = p < 0.001, green = p < 0.01, yellow = p < 0.05, blue = p > 0.05 (not significant)

Fig. 5

PAE females display increased cortical porosity in the posterior region of the tibiofibular junction. (A) 3D renderings of intracortical canals (red) from male and female control and PAE mice. (B) Percentage cross-sectional cortical porosity and (C) percentage regional cortical porosity in anterior, medial, lateral and posterior regions. Data are presented as mean ± SEM with points showing individual animals. # denotes significant difference from anterior region within experimental group; ¤ denotes significant difference from medial region within experimental group; † denotes significant difference from lateral region within experimental group; * denotes significant difference between specific regions in different experimental groups. One symbol = p < 0.05, two symbols = p < 0.001, three symbols = p < 0.001

Fig. 6

PAE decreases growth plate width in male mice. (A) TRAP and haemotoxylin and eosin-stained sections of male and female PAE and control 12-week-old mice. (B) Total growth plate zone width, (C) Proliferative zone width, and (D) Hypertrophic zone width, of male and female control and PAE mice. Ten measurements per section were obtained along the length of the tibial growth plate in the middle region of the knee joint. (E) Location and areal densities of bridges across the growth plate projected on the tibial joint surface in male and female control and PAE mice. (F) Number of bridges. (G) Areal density (d) of bridges defined as the number of bridges per 256 mm x 256 mm window. Data are presented as mean ± SEM with points showing individual animals. *= p < 0.05, * p < 0.01, ***= p < 0.001

Fig. 7

Juvenile mice do not display the altered skeletal phenotype that are observed in skeletally mature mice. µCT analysis of the trabecular compartment, including (A) tissue volume, (B) bone volume, (C) bone volume to tissue volume ratio (BV/TV), (D) bone surface, and (E) trabecular number (Tb.N), (F) trabecular pattern factor (Tb.Pf), (G) fractal dimension, and (H) connectivity density (Conn.Dn) in control and PAE males and females. Data are presented as mean ± SEM with points showing individual animals. *= p < 0.05, ***= p < 0.001, ****= p < 0.0001. Measurement and statistical analysis heat map for (I) tissue area (T. Ar), (J) tissue perimeter (T.Pm), (K) Bone area (B.Ar), (L) resistance to torsion (J), (M) maximum second moments of inertia (I_max) and (N) minimum second moments of inertia (I_min) in male and female PAE and control mice. Line graphs represent mean ± SEM for male control (black), male PAE (orange), female control (green) and female PAE (blue) mice. Graphical heat map summarises statistical differences at specific matched locations along the tibial length (10–90%) for the PAE effect in females (Ctl-PAE F) and males (Ctl-PAE M), and the sex effect in control (F-M Ctl) and PAE (F-M PAE) groups. Red = p < 0.001, green = p < 0.01, yellow = p < 0.05, blue = p > 0.05 (not significant)

Fig. 8

Sexually dimorphic effects of PAE on the skeleton. PAE osteoblasts from males and females produce less bone nodules than normal cells yet gene expression is differentially affected. This has lasting effects on the skeleton as PAE in 12-week-old male mice detrimentally alters the trabecular and cortical regions of the bone, bone mechanical properties and the growth plate. However, PAE female mice are protected in the growth plate and trabecular regions, but show detrimental effects only in the distal region of the tibia and at the tibiofibular junction