Diet-induced obesity influences oocyte developmental competence via peroxisome proliferator-activated receptor gamma (PPARG)-mediated mechanisms.

Abstract

Across the world more women of childbearing age are becoming overweight and obese. Although overweight women have similar co-morbidity and stigmata as men they also experience problems specific to their gender. In particular, there is significant evidence that overweight and obese women require a longer time to successfully conceive, suggesting influence of bodyweight and adipose tissue mass upon the events surrounding conception. This thesis investigated the interaction between diet-induced obesity and female reproductive function. To achieve this, the influence of maternal obesity-induced insulin resistance on ovulation and oocyte health, as indicated by subsequent embryonic developmental competence was determined. Obesity adversely affects many aspects of health, and rodent models of diet-induced obesity are commonly used to investigate these consequences. However the impact of strain and genetic background on phenotypic response to diet, particularly in females, has not been systematically defined. We therefore characterised female metabolic responses of five different strains of laboratory mouse (Swiss, Balb/c, C57BL/6, CBA/CaH and 129T2Sv/Ems) to a “Western” high fat diet (22% fat, 0.15% cholesterol) and matched control diet (6% fat, 0% cholesterol). After 16 weeks of diet exposure the development and extent of hyperglycaemia, hyperinsulinaemia, insulin resistance, dyslipidaemia, and markers of chronically inflamed adipose tissue depots varied profoundly across the different strains. To then determine if a perturbed metabolic profile triggers female infertility, these female mice were mated with strain matched, non-obese males, and zygotes extracted from the reproductive tract immediately following fertilization. Despite strain-dependent variation in susceptibility to the development of obesity, dyslipidaemia and insulin resistance, all mice investigated exhibit some degree of impaired reproductive potential following exposure to a high fat diet. We documented alteration to ovulation incidence and rate, fertilization, early embryo development to the blastocyst stage, and blastomere differentiation into the inner cell mass and trophectoderm cell lineages. The nature of obesity-induced perturbation of female reproductive processes was more closely examined using statistical modelling which identified the specific metabolic parameters that were strongly associated with reproductive defects. These associations were consistent across the range of genetic backgrounds assessed and highlighted key mediators of this interaction, in particular, insulin resistance. To determine if ovarian gene products already implicated in other reproductive outcomes are differentially regulated under conditions of obesity, ovarian mRNA collected at the pro-estrous (preovulatory) stage of the reproductive cycle was applied to microarray slides developed through Suppressive Subtractive Hybridization. Two different gene chips that were enriched for ovarian genes were used. A number of genes were minimally regulated, and there was lack of significant validation in subsequent, and larger, sample cohorts. These findings have provided substantial technical information, and new experimental designs that overcome the current limitations have been established to obtain more informative data. The role that insulin resistance plays in folliculogenesis and the development of oocyte developmental competence was more closely investigated. Hyperinsulinemia can interfere directly with ovarian cell function or be indirectly associated with other hormonal conditions detrimental to optimal fertility. To reverse the effects of obesity/hyperinsulinemia and identify the signalling pathways responsible for disruption of pre-implantation events, obese female mice were treated for 4 days prior to mating with three different insulin-sensitizing and plasma glucose-reducing pharmaceuticals: glucose and lipidlowering AMP Kinase activator, AICAR, 30mg/kg/day; IκK inhibitor that reverses insulin resistance, sodium salicylate, 50mg/kg/day; or Peroxisome Proliferator-Activated Receptor Gamma (PPARG) agonist rosiglitazone, 10mg/kg/day. AICAR or sodium salicylate treatment did not have significant effects on the reproductive parameters examined. However, embryonic development to the blastocyst stage was significantly improved when diet-induced obese mice were treated with rosiglitazone, effectively repairing development rates. Rosiglitazone also normalized obesity-associated abnormal blastomere allocation to the inner cell mass. Such improvements to oocyte quality were coupled with weight loss, improved glucose metabolism and changes in ovarian mRNA expression of PPARGregulated cholesterol transporters. Overall, this thesis has demonstrated for the first time a link between maternal obesity and the ovarian follicle can impede oocyte health and developmental potential. As a result, the oocyte released at ovulation expresses impaired developmental competence following to conception. Key cellular pathways have been identified in this relationship, specifically PPARG-directed cell responses.