Cytokines and programming the pre-implantation embryo.

Abstract

As the pre-implantation embryo traverses the female reproductive tract, it experiences fluctuations in the composition of the surrounding maternal environment, including the availability of nutrients, growth factors and cytokines. In particular, the cytokine milieu surrounding the early embryo is pivotal in programming optimal embryo development. The pre-implantation embryo is sensitive to a range of perturbations such as maternal diet or in vitro culture. These and other insults influencing the maternal environment including infection, stress and environmental toxins may in part act via impact on oviduct and uterine cytokine synthesis. However the effect of maternal perturbation to inflammation or infection, on the embryo and the role of cytokines in mediating this is not fully elucidated. The studies described in this thesis employed an in vivo mouse model of maternal systemic inflammation with the proinflammatory bacterial lipopolysaccharide (LPS), where a pro-inflammatory cytokine response was elicited on days 2.5 and 3.5 post coitum (pc), prior to implantation. This model was studied in wildtype C57Bl/6 (Il10 ⁺ʹ⁺) mice and mice with a null mutation in the Il10 gene (Il10 ⁻ʹ⁻) were studied to investigate the effects of maternal deficiency in the anti-inflammatory cytokine IL-10 during LPS treatment. We demonstrated that the altered cytokine signals resulting from a low level pro inflammatory LPS challenge (0.5 μg/mouse) in the pre-implantation period elicit changes in the embryo developmental trajectory that in turn alter fetal growth and delay postnatal growth in the male progeny from LPS-treated mothers. As LPS did not directly impact development of the embryo at low and moderate doses, this result appears to reflect indirect effects of LPS mediated via the maternal tract. This is consistent with data from day 3.5 pc oviduct and uterus tissues which revealed increased mRNA expression of proinflammatory cytokines including Il6, Tnfa and Il12b following maternal LPS treatment. Peri-conceptional low dose LPS treatment in Il10 ⁺ʹ⁺ and Il10 ⁻ʹ⁻ mice revealed that the number of viable fetuses and fetal weight were both significantly reduced after LPS treatment, particularly in the Il10 ⁻ʹ⁻ mice. Embryo transfer was then utilised to investigate the mechanism by which LPS acts on the embryo, where day 3.5 pc embryos from donors treated with 0.5 μg LPS or PBS on days 2.5 and 3.5pc were transferred into day 2.5 pc pseudopregnant Swiss female recipients. The effect of maternal LPS treatment on fetal and placental development was seen to be maintained even after embryo transfer, suggesting that any effects of altered cytokine expression in embryos are exerted during cleavage stages before embryo recovery from donors. In addition, postnatal investigation of male and female progeny derived from control PBS and LPStreated Il10 ⁺ʹ⁺ and Il10 ⁻ʹ⁻ females from birth until 19 weeks of age showed that maternal LPS treatment constrains postnatal growth in male progeny regardless of maternal Il10 genotype, compared to male progeny from PBS-treated mothers. While the adult male progeny from LPS-treated Il10 ⁺ʹ⁺ and Il10 ⁻ʹ⁻ mothers did not display changes in fat mass compared to their PBS-treated control counterparts, the combination of maternal LPS treatment and maternal IL-10 deficiency resulted in greater fat mass accumulation in the adult male progeny from LPS-treated Il10 ⁻ʹ⁻ mothers compared to adult male progeny from LPS-treated Il10 ⁺ʹ⁺ mothers. In addition, we investigated the effects of maternal systemic inflammation during the pre-implantation period on the response to LPS challenge during adulthood. Male progeny from LPS-treated Il10 ⁻ʹ⁻ mothers had a dampened response in LIF cytokine following a 100μg/kg LPS challenge at 18 weeks of age. This study implies a role for cytokines as mediators of programming the embryo during the preimplantation period, with altered responses in the event of maternal systemic inflammation impacting on later fetal and postnatal development. The anti-inflammatory cytokine IL-10 acts to protect the embryo from the adverse programming effects of exposure to LPS during the pre-implantation period, with absence of IL-10 resulting in altered postnatal phenotype and particularly fat mass accumulation in the male progeny during adulthood. It appears likely that the absence of IL-10 in the maternal environment delays the clearance of adverse pro-inflammatory cytokines induced during an inflammatory challenge, resulting in prolonged exposure of the embryo to circulating pro-inflammatory cytokines in the maternal tract, supporting a cytokine-mediated mechanism. These studies provide additional evidence for a role of cytokines in embryo sensing of environmental conditions, and indicate that IL-10 is a key regulator of this communication pathway.