Figure 4. Kiss1^Cre:Nhlh2^fl/fl male mice present delayed puberty onset while females display irregular estrous cycles.

Female Kiss1^Cre:Nhlh2^fl/fl mice present normal puberty onset as assessed by daily monitoring of vaginal opening (VO) and documented by cumulative percent of animals at VO (A), mean age of VO (B) Nhlh2^fl/fl (n=15) and Kiss1^Cre:Nhlh2^fl/fl (n=10) and first estrus (C) Nhlh2^fl/fl (n=14) and Kiss1^Cre:Nhlh2^fl/fl (n=9). These females have normal body weight (BW) at the age of VO (D). Estrous cyclicity, assessed by daily vaginal cytology for 30 days is irregular in Kiss1^Cre:Nhlh2^fl/fl mice (E, F), presenting longer time in diestrus compared to control, Nhlh2^fl/fl (n=8) and Kiss1^Cre:Nhlh2^fl/fl (n=9). *p<0.05 two-way ANOVA followed by Bonferroni test. (G) LH measurements in the morning (AM [10 a.m.]) and afternoon (PM [7:30 p.m.]) after lights off of female mice subjected to an LH surge induction protocol. n=6/group. Groups with different letters are significantly different. Two-way ANOVA followed by Bonferroni post hoc test. (H, I) Pubertal development of male Kiss1^Cre:Nhlh2^fl/fl mice and control littermates determined by preputial separation. *p<0.05 by Student's t-test. (J) BW of males at the age of preputial separation. Nhlh2^fl/fl (n=19) and Kiss1^Cre:Nhlh2^fl/fl (n=9). *p<0.05 by Student's t-test. Data presented as median (dotted line)+distribution of the data and its probability density (violin plot). cKO=Kiss1^Cre:Nhlh2^fl/fl. LH, luteinizing hormone.

Figure 4—figure supplement 1. Validation of the Kiss1^Cre:Nhlh2^fl/fl mouse model.

Representative images depicting the absence of co-expression of Kiss1 (green) and Nhlh2 (red) mRNA in the ARH of adult Kiss1^Cre:Nhlh2^fl/fl male mice (A). White arrows indicate examples of Kiss1 neurons showing the absence of Nhlh2 expression in them. Expression of Nhlh2 in testes (B) and ovaries (C) from adult Kiss1^Cre:Nhlh2^fl/fl mice and their littermate controls (n=4–5/group). cKO=Kiss1^Cre:Nhlh2^fl/fl.

Figure 4—figure supplement 2. Kiss1^Cre:Nhlh2^fl/fl mice display normal body weight (BW).

BW of Kiss1^Cre:Nhlh2^fl/fl male and female mice and their respective littermate controls were monitored for 130 days under regular chow diet followed by 11 weeks on a 60% high-fat diet (HFD). cKO=Kiss1^Cre:Nhlh2^fl/fl.