Maternal Adaptation to Pregnancy: Altered Circadian Rhythms of Clock Genes in the Hypothalamus and Metabolic Tissues

Maternal physiological adaptations are crucial for pregnancy success by ensuring that fetal and placental demands are met whilst maintaining maternal homeostasis. There is growing recognition that whole body homeostasis is intimately linked to circadian variation. Accordingly, major physiological adaptations in pregnancy likely involve marked shifts in maternal circadian function. Circadian variation of physiological processes are driven by a network of clock genes that form transcriptional-translational feedback loops in both central (i.e. the suprachiasmatic nucleus; SCN) and peripheral tissues including the liver and adipose tissue. Therefore, the aims of this thesis were to characterise the impact of pregnancy on clock gene expression in key maternal tissues involved in HPA axis regulation and glucose and lipid metabolism as well as to determine subsequent downstream effects on the expression of tissue-specific genes that are known to be regulated in a circadian manner. The initial study in this thesis tested the hypothesis that hypothalamic expression of clock genes change across mouse pregnancy and that this is linked to altered HPA axis activity. The anterior hypothalamus and plasma were collected from C57Bl/6J mice prior to and on days 6, 10, 14 and 18 of pregnancy (term=day 19) at 4-h intervals across a 24-h period. Hypothalamic expression of all clock genes changed markedly across gestation, particularly at mid-gestation when levels of each gene were elevated. The pregnancy-induced increase in maternal corticosterone (up to 14-fold higher by day 14) was not accompanied by a parallel shift in plasma adrenocorticotropic hormone (ACTH; 28% lower on day 14 compared with non-pregnant). Moreover, while circadian rhythmicity in corticosterone was maintained up to day 14, this was effectively lost by day 18. Thus, the central circadian clock undergoes marked adaptations throughout pregnancy, changes that likely contribute to maternal physiological adaptations. Importantly, however, neither hypothalamic clock genes nor plasma ACTH appear to drive the late-gestational increase in maternal corticosterone. The second study tested the hypothesis that maternal adaptation to pregnancy involves changes in the hepatic expression of clock genes, which drive downstream shifts in the circadian expression of glucoregulatory genes. Maternal liver was collected at the same time-points as described for the initial study. Hepatic clock gene expression was substantially altered across pregnancy, most notably in late gestation when the circadian Pregnancy-induced adaptations of maternal tissue clocks iv rhythmicity of several clock genes was attenuated (e.g. 64% reduced amplitude for Bmal1 on day 18). Similar reductions were observed in rhythmicity of the key glucoregulatory genes phosphoenolpyruvate carboxykinase 1 (Pck1), glucose-6phophatase and glucokinase, and by day 18 Pck1 was no longer rhythmic. Thus, marked adaptations in the liver clock occur during mouse pregnancy and may contribute to the altered circadian variation in glucoregulatory genes near term. We propose that the reduction of daily oscillations in glucose metabolism ensure a sustained supply of glucose to meet high fetal demands. The third study hypothesised that pregnancy alters clock gene and downstream metabolic gene expression in white adipose tissue. Gonadal adipose tissue was collected from pregnant mice at the time-points described above. Adipose clock gene expression varied substantially across gestation, although the robust rhythmicity of expression was maintained throughout. In contrast, the circadian expression of the metabolic genes hormone sensitive lipase, adipocyte triglyceride lipase and lipoprotein lipase were lost with pregnancy onset. These findings demonstrate significant gestational adaptations in the adipose clock and a pregnancy-induced dissociation of adipose lipolytic and lipogenic genes from the circadian regulation of adipose clock genes during normal pregnancy. Changes in the rhythmicity of maternal core body temperature (Tc) across pregnancy were also investigated as a possible mechanism driving pregnancy-induced changes in adipose clock gene expression. Tc was measured throughout gestation by intraperitoneal temperature loggers. Tc rhythm showed a 0.61°C decrease in mesor between days 6 and 18 and a 65% decrease in amplitude by term. These changes, however, were not linked to adipose clock gene expression during pregnancy. Overall, these studies demonstrate for the first time that clock gene expression is markedly altered in key maternal tissues throughout normal mouse pregnancy. Furthermore, the effect of pregnancy on clock gene and downstream gene expression is highly tissue-dependent. In conclusion, pregnancy-induced changes in the circadian oscillations of both clock genes and metabolic genes are substantial and thus should be considered part of the normal maternal physiological adaptation to pregnancy. Pregnancy-induced adaptations of maternal tissue clocks v