Sex dependent intergenerational effects of lead in mouse model

Abstract

Lead (Pb) exposure negatively impacts fertility in both males and females, pregnancy outcomes, and child brain development. We investigated the reproductive and neurological effects of Pb exposure on male and female mice via Pb-contaminated soil for 4 weeks. Breeding was conducted after completion of exposure, in four groups; group 1 consisted of exposed dams and unexposed sires, group 2 consisted of exposed sires and unexposed dams, group 3 consisted of exposed sires and exposed dams and group 4 was the control. Generally, Pb exposure reduced observed conception rates, with a cumulative decrement observed when both males and females are exposed. Gene expression of the testes revealed oxidative stress as the cause of reduced conception rates. Neurological tests: Morris water maze and rotarod were conducted on F1 generation offspring. Maternally and paternally exposed F1 mice performed poorly in the Morris water maze when compared to the control. The severity of the neurological effects was also parent-dependent and sex-dependent. Paternal Pb exposure effects were more pronounced in female offspring. A comparison of gene expression changes of the hippocampus and prefrontal cortex showed paternal Pb-exposure resulted in more prefrontal cortex changes than in the hippocampus, a trend also recorded in the exposed sires. The pronounced effects in female offspring of paternal Pb exposure may suggest that Pb neurological effects may be X-chromosome-linked.

Keywords: Infertility; Maternal; Neurological; Paternal; Pb; Soil.

Fig. 1

Shows the timeline of the animal exposure experiment.

Fig. 2

Shows lead levels in the bone and blood of F0. (A) Shows blood Pb levels and (B) shows bone Pb levels in F0 sires. (C) Shows blood Pb levels and (D) shows bone Pb levels in F0 dams. *Indicates p < 0.01.

Fig. 3

Shows lead levels in the bone and blood of pregnant and non-pregnant dams of the exposed group. (A) Shows the bone lead levels and (B) shows blood lead levels in exposed dams that were able to get pregnant and those that did not at weaning. *P-value = 0.04.

Fig. 4

Shows latency to escape and distance covered in the Morris Water Maze of F1 females. (A) Shows average latency to escape during training of Water Morris Test and (B) shows distance during latency to escape during training of Water Morris Test in F1 females. (n = 6 in each group).

Fig. 5

shows latency to escape, and distance covered in F1 males during the water Morris maze test. (A) Shows latency to escape during training period. (B) Shows distance travelled during training period in F1 males. (n = 9 in each group).

Fig. 6

Shows the results of the probe test for F1 females and F1 males. (A,D) Shows latency to escape during probe test (*p-value = 0.01), (B,E) shows entries into target zone and (C,F) shows time spent in target zone in F1 females and F1 males, respectively. (n = 6 in each group in F1 females and n = 9 in each group in F1 males).

Fig. 7

Shows rotarod test results for F1 females and F1 males. (A,C) Shows speed attained during rotarod test, (B,D) shows total time spent on rotarod test by F1 females and F1 males respectively. (*P- value < 0.05). (n = 6 in each group in females and n = 9 in each group in males).

Fig. 8

shows lead levels in the bone and blood of F1 Females and F1 males. (A,B) Represent bone lead levels at 4 weeks old in F1 females and males respectively, while (C,D) are the bone lead levels at 9 weeks old. (n = 3 in each group at 4 weeks in both F1 females and F1 males), (At 9 weeks n = 6 in each group in females and n = 9 in each group in males). *p-value = 0.03. The Both exposed group had only 7 females at weaning and hence none were culled at 4 weeks old and Pb concentrations are not presented.

Fig. 9

shows fertility and oxidative stress associated genes expression levels in sire testicular tissue. (A) Shows Pou5F1 gene expression, (B) shows Crisp2 expression, (C) shows Spink2, (D) shows Nrf2 expression in the testicular tissue of F0 sires. *p-value = 0.02. Sires’ brain expression genes and Keap1 are shown in the supplementary material.

Fig. 10

shows expression levels of brain plasticity related genes in F1 females. (A) Bdnf expression, (B) shows Creb expression, (C) shows Grin2A expression and (C) shows Grin2B expression in F1 females. *Indicates p-value = 0.03, **indicates p-value = 0.001. (n = 6 in each group).

Fig. 11

shows expression levels of plasticity associated genes in F1 females in the hippocampus. (A) Shows Bdnf expression levels, (B) shows Creb expression, (C) shows Grin2A expression and (D) shows Grin2B expression in F1 males. (n = 6 in each group).

Fig. 12

shows expression levels of plasticity associated genes in F1 males. (A) Shows Bdnf expression levels, (B) shows Creb expression, (C) shows Grin2A expression and (D) shows Grin2B expression in F1 males. (n = 6 in each group).

Fig. 13

shows expression levels of plasticity associated genes in F1 females in the hippocampus. (A) Shows Bdnf expression levels, (B) shows Creb expression, (C) shows Grin2A expression and (D) shows Grin2B expression in F1 males. (n = 6 in each group).

Fig. 14

(A) Shows a heatmap of gene expression comparison between hippocampus and prefrontal cortex in F1 females, where C represents prefrontal cortex and H for hippocampus. (B) Shows a heatmap of gene expression comparison between hippocampus and prefrontal cortex in F1 males, where C represents prefrontal cortex and H for hippocampus.

Fig. 15

Shows a schematic diagram that describes relationship between Bdnf, Glu2B, Glu2A and their corresponding genes.

Fig. 16

Demonstrates a flow chart of how intergenerational lead exposure leads to sex-linked neurological deficits depending on which parent is exposed.