The role of the anterior and ventromedial hypothalamic regions in mediating leptin's effect on fertility

Abstract

Reproductive function is an energetically demanding process and is therefore tightly regulated by peripheral energy status. This is illustrated by delayed puberty onset and impaired adult reproductive function in individuals with a state of suboptimal energy availability. Furthermore, obesity and its related metabolic syndromes are also associated with impaired adult reproductive function. It is well established that the adipocyte-derived hormone leptin plays an essential role in the regulation of reproductive function, as leptin signalling in the hypothalamus is required for normal sexual maturation and adult fertility. Leptin indirectly stimulates gonadotropin releasing hormone (GnRH) neurons via several afferent neuronal populations, the interaction of often occurs in a sexually dimorphic manner. Recent evidence suggests an important role for leptin actions via neurons located in the preoptic area. The initial aim of this project was to elucidate whether preoptic neurons are sufficient to mediate leptin signalling; permitting normal reproductive function in the absence of leptin signals from any other region. Cre-lox methodology was used to generate mice in which expression of leptin receptor (LepR) was Cre-dependent. In adulthood, a proportion of these underwent selective activation (‘rescue’) of LepR only in the area by stereotaxic unilateral injection of an adeno-associated virus (AAV) containing Cre DNA. This enabled comparison of puberty onset and adult fertility between LepR-rescue, LepR-null and wild-type (WT) mice. Puberty onset was indicated by preputial separation in males and by vaginal opening and first estrus in females. Post-AAV analysis of sexual maturation and restoration of steroid hormone production was assessed by reproductive cyclicity and uterine weight in females and by reproductive organ weight in males. Adult fertility was assessed by circulating levels of luteinising hormone (LH) and fecundity. Analysis of the leptin signalling molecule pSTAT3 revealed the LepR rescue occurred further caudally than the intended target (the medial preoptic area); rather, it was localised to the anterior hypothalamic area (AHA) and ventromedial nucleus (VMH). As expected, 100% of WT and 0 female LepR-null mice underwent puberty onset prior to 2 months of age. However, 2-4 weeks following AAV treatment, 60% of female LepR-rescue mice had puberty onset, including restored reproductive cycles, compared to 16% of LepR-null mice (p=0.19). Further, female LepR-rescue mice exhibited significantly increased uterine weight (p=0.04) as well as a trended higher luteinising hormone concentration post-AAV treatment compared to LepR-null mice. LepR rescue in males did not significantly improve puberty onset or indices of sexual maturation. These results suggest leptin signalling only in the AHA and VMH may be sufficient for reproductive function in females. Interestingly, the AHA is not known to be important for reproductive function. Although the exact leptin-responsive neurons causing these results cannot be determined, further investigation into the AHA is warranted. Of particular interest is the recent evidence to support a female-specific role for the VMH in the mediation of leptin’s permissive effect on GnRH neurons. An enhanced understanding of the specific mechanism by which leptin regulates reproductive function is imperative to the pursuit of improved fertility in a world that is severely burdened by both undernutrition and obesity, and in which many couples are unable to conceive.