The morphological adaptations of the maternal liver to pregnancy in the mouse

Abstract

The liver is known to increase in size in the mouse during pregnancy; however, the mechanisms behind this are not fully understood. There are inconsistencies in the literature, which argue whether hyperplasia or hypertrophy is responsible. The aim of this thesis was to investigate the possible mechanisms and to explain some of the molecular processes, which bring these adaptations about.

To address this, virgin female C57Bl/6 mice were mated and culled at 3 time points during pregnancy and their livers analysed. Non-pregnant, age matched mice were analysed as controls. Phenotypic measurements of the mice were taken in order to make sure the model represented known changes seen during mouse pregnancy.

Haemotoxylin and eosin staining showed that there is an increase in cell size from day 14 of gestation. Staining for Ki67, a cell cycle marker, suggested that cell proliferation was also increased at day 14 and day 18 of gestation. This was further supported by gene expression analysis of Ki67 and cyclin B1.

Transcriptome analysis was carried out on the livers at both day 14 and day 18 of gestation. Pathway analysis predicted an increase in cell proliferation at both time points and a decrease in apoptosis at day 18 of gestation. These pathways were further validated by analysis of a previously published array data set, showing the same pathway changes at all time-points measured. Gene expression of the apoptosis markers p53, Fasand Casp6 were used to strengthen the evidence for down regulation of apoptosis in pregnancy. Fas and p53 expression was supressed at all pregnancy time points measured and Casp6 was down regulated at both day 14 and day 18 of gestation.

It was hypothesised that changes in expression may be regulated by changes to DNA methylation. Global methylation measurements and expression of DNA methylation associated enzymes (Dnmt1, 3a, 3b, Tet1, 2 and 3) initially suggested a decrease during pregnancy, supporting this hypothesis. However, methylated DNA immunoprecipitation sequencing showed that there were only 6 differentially methylated regions in the liver during pregnancy and only one of these corresponded to a change in gene expression. This suggests that DNA methylation was unlikely to be the mechanism responsible for the changes in gene expression observed.

It was thought that the changes in the mouse liver during pregnancy might be brought about by exposure to pregnancy-associated hormones. To test this, mouse Hepa1-6 liver cells were exposed to a range of concentrations of oestrogen, mouse prolactin, human prolactin and human placental lactogen. No change was found in global DNA methylation and the expression of the Dnmt and Tet genes showed large variation. However, treatment with specific concentrations of the hormones reduced the variation seen in expression in these genes. As Dnmt expression has been shown to vary with the cell cycle, it was hypothesised that exposure of the cells to hormones at these concentrations led to an alignment of the cell cycle and, therefore, reduced the variation in expression. Flow cytometry was used to identify the proportion of cells at each stage of the cell cycle. It was found that treatment with human prolactinreduced the proportion of apoptotic cells and altered the distribution of cells at other points in the cell cycle.

Having observed the response of the mouse liver to normal pregnancy, showing increased proliferation and decreased apoptosis, it was hypothesised that changing the maternal diet may disrupt this response. Female mice were fed increasing fat content diets (with reciprocal decrease in carbohydrate) and the expression of Ki67, cyclinB1, Fas, p53 and Casp6 measured. There were no significant changes in the expression of these genes.

Phenotypic analysis of the mouse liver showed that both hypertrophy and hyperplasia play a role in the increase in size seen during pregnancy. Gene expression analysis added strength to this, as well as showing that a reduction of apoptosis provides an additional mechanism suggesting how this tissue growth is maintained. Use of an in vitro cell model showed that stimulation with prolactin may cause the decrease in apoptosis and at least contribute to the stimulation of cell proliferation.

Together, these findings provide novel insights into the mechanisms that underlie the expansion of the mouse liver in pregnancy. The findings may also have implications in identifying targets for treatment in diseases such as hepatocellular carcinoma and obstetric cholestasis.

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Identifiers

ORCID for Graham Burdge: orcid.org/0000-0002-7665-2967

ORCID for Karen Lillycrop: orcid.org/0000-0001-7350-5489

ORCID for Mark Hanson: orcid.org/0000-0002-6907-613X

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