Polycystic ovary syndrome (PCOS) is a common endocrine disorder in reproductive-aged women that is comprised of two out of the following three features: hyperandrogenism, oligo- or amenorrhea, or polycystic ovaries. In addition to infertility, many women with PCOS have metabolic dysregulation that increases the risk of developing type 2 diabetes, hypertension, and non-alcoholic fatty liver disease. Changes in the gut microbiome are associated with PCOS and gut microbes may be involved in the pathology of this disorder. Since PCOS often manifests in the early reproductive years, puberty is considered to be a critical time period for the development of PCOS. Exposure to sex steroid hormones during development results in permanent, organizational effects, while activational effects are transient and require the continued presence of the hormone. Androgens exert organizational effects during prenatal or early post-natal development, but it is unclear whether androgen excess results in organizational or activational effects during puberty. We recently developed a letrozole-induced PCOS mouse model that recapitulates both reproductive and metabolic phenotypes of PCOS. In this study, we investigated whether letrozole treatment of pubertal female mice exerts organizational or activational effects on host physiology and the gut microbiome. Two months after letrozole removal, we observed recovery of reproductive and metabolic parameters, as well as diversity and composition of the gut microbiome, indicating that letrozole treatment of female mice during puberty resulted in predominantly activational effects. These results suggest that if exposure to excess androgens during puberty leads to the development of PCOS, reduction of androgen levels during this time may improve reproductive and metabolic phenotypes in women with PCOS. These results also imply that continuous letrozole exposure is required to model PCOS in pubertal female mice since letrozole exerts activational rather than organizational effects during puberty.

Stress suppresses pulsatile luteinising hormone (LH) secretion in a variety of species, although the mechanism underlying this inhibition of reproductive function remains unclear. Metabolic stress, particularly hypoglycaemia, is a clinically-relevant stress type that is modelled with bolus insulin injection (insulin-induced hypoglycaemia). The present study utilised ovariectomised C57BL/6 mice to test the hypothesis that acute hypoglycaemia suppresses pulsatile LH secretion via central mechanisms. Pulsatile LH secretion was measured in 90-minute sampling periods immediately prior to and following i.p. injection of saline or insulin. The secretion of LH was not altered over time in fed animals or acutely fasted (5 hours) animals following an i.p. saline injection. By contrast, insulin elicited a robust suppression of pulsatile LH secretion in fasted animals, preventing LH pulses in five of six mice. To identify the neuroendocrine site of impairment, a kisspeptin challenge was performed in saline or insulin pre-treated animals in a cross-over design. LH secretion in response to exogenous kisspeptin was not different between animals pre-treated with saline or insulin, indicating normal gonadotrophin-releasing hormone cell and pituitary responses during acute hypoglycaemia. Based on this finding, the effect of insulin-induced hypoglycaemia on arcuate kisspeptin (Kiss1) cell function was determined using c-Fos as a marker of neuronal activation. Insulin caused a significant suppression in the percentage of Kiss1 cells in the arcuate nucleus that contained c-Fos compared to saline-injected controls. Taken together, these data support the hypothesis that insulin-induced hypoglycaemia suppresses pulsatile LH secretion in the female mouse via predominantly central mechanisms, which culminates in the suppression of the arcuate Kiss1 population.

Background

A majority of women with polycystic ovary syndrome (PCOS) have metabolic dysfunction that results in an increased risk of type 2 diabetes. We previously developed a pubertal mouse model using the aromatase inhibitor, letrozole, which recapitulates many of the reproductive and metabolic features of PCOS. To further our understanding of the effects of androgen excess, we compared the effects of letrozole treatment initiated in puberty versus adulthood on reproductive and metabolic phenotypes as well as on the gut microbiome.

Results

Letrozole treatment of both pubertal and adult female mice resulted in reproductive hallmarks of PCOS, including hyperandrogenemia, anovulation and polycystic ovaries. However, unlike pubertal mice, treatment of adult female mice resulted in modest weight gain and abdominal adiposity, minimal elevation in fasting blood glucose and insulin levels, and no detectable insulin resistance. In addition, letrozole treatment of adult mice was associated with a distinct shift in gut microbial diversity compared to letrozole treatment of pubertal mice.

Conclusions

Our results indicate that dysregulation of metabolism and the gut microbiome in PCOS may be influenced by the timing of androgen exposure. In addition, the minimal weight gain and lack of insulin resistance in adult female mice after letrozole treatment indicates that this model may be useful for investigating the effects of hyperandrogenemia on the hypothalamic-pituitary-gonadal axis and the periphery without the influence of substantial metabolic dysregulation.