A novel leptin receptor mutation causes delayed puberty onset in mice

Reproductive function is tightly controlled by nutritional state due to its high energetic demands. Regulation of fertility according to available energy stores is attained through the integration of metabolic status signals from the periphery with the hypothalamic pituitary gonadal (HPG) axis, which governs reproductive function. The hormone leptin plays an integral role in the normal functioning of the reproductive system due to its ability to communicate energy status (i.e. stored fat reserves) to the brain’s reproductive centres. Mutations in leptin or its receptor which cause impairment in signalling have been previously characterized to cause infertility and morbid obesity in mice. Recently, Juengel et al and Haldar et al identified two naturally occurring leptin receptor (LepR) mutations (R62C and P1019S) in Davisdale sheep which were associated with a significant delay in puberty onset and poor ovulation rates. However, it is unknown whether these mutations cause the same fertility deficits in other species. This project aimed to determine whether these two naturally occurring LepR mutations are associated with defects in puberty onset and adult fertility in mice. CRISPR-Cas gene editing was used to create two mouse lines with ‘knock-in’ leptin receptor (LepR) mutations (A63C and P1018S, resulting in identical amino acid substitutions as the sheep mutations). For each of the two mouse lines, wildtype, heterozygous, and homozygous mutant males and females (n=8-14 per group) were identified using polymerase chain reaction (PCR) and agarose gel genotyping. Puberty onset was measured post-weaning by daily visual examination of the genitalia. In males, this was indicated by preputial separation, and in females by vaginal opening followed by onset of estrous (reproductive) cycles. Adult fertility and steroid hormone production were assessed by reproductive cyclicity in females and sex steroid-sensitive reproductive organ weight and breeding success in both sexes. The metabolic effects of these LepR mutations were assessed via body weight and abdominal fat weight measurements. Analysis of puberty onset revealed that A63C homozygous and heterozygous males had significantly delayed (by 4 [p=0.008] and 5 [p=0.0011] days, respectively) puberty compared to their wildtype counterparts (Kruskal-Wallis test with Dunn’s post-hoc test). A63C homozygous and heterozygous females also showed significantly delayed (by 4 [p=0.02] and 5 [p=0.017] days, respectively) first estrus compared to their wildtype counterparts (Kruskal-Wallis test with Dunn’s post-hoc test). This result demonstrates that the A63C LepR mutation has the ability to cause delayed puberty in other species, which could warrant further investigation into similar human LepR mutations. If the mutation is also associated with later-life subfertility, this study could contribute to potential downstream clinical applications including new and improved diagnoses of genetic risk factors, as well as treatments to address the high rate of human infertility