Article Figures & Data
Figures
- Figure 1.
Strategy for knocking TH exclusively out of Kiss1 cells. A, The map of the TH “floxed” gene insert in embryonic stem cells (KOMP repository, UC Davis). Presence of the neomycin cassette in PCR analysis was indicative of germline transmission of the TH flox insert. B, Breeding neomycin positive females to Flp recombinase males removed the lacZ and neomycin cassettes and produced TH flox heterozygous mice. These TH fl/wt mice were then bred to each other to produce TH fl/fl mice. C, KissCre mice were bred to TH fl/fl mice to produce KissCre+ TH fl/fl mice (Kiss THKOs), which lack exons 7-9 of the TH gene and thus, a functional TH transcript, but only in Kiss1 cells. D, Gel image of PCR analysis showing Cre-mediated recombination of TH, indicated by the 503 bp band representing successful recombination of the TH gene, only in tissues known to express Kiss1 (AVPV/PeN, ARC, MeA, liver, gonad) and not in other tissues (cerebellum, spleen, tail), which are known to not normally express Kiss1 in Kiss THKO (top) and WT (bottom) mice. E, left, Photomicrograph of TH (silver grains) and Kiss1 (red fluorescence) double label ISH in the AVPV/PeN region of adult female mice. TH mRNA is clearly highly expressed in almost all AVPV/PeN Kiss1 neurons (examples indicated by yellow arrows) of WT mice but absent in virtually all Kiss1 cells in Kiss THKO mice. Right, Quantification of the percentage of AVPV/PeN Kiss1 cells coexpressing TH in WT and KissTHKO adult female mice (n = 4-5/genotype).
- Figure 2.
Removal of TH selectively from just Kiss1 neurons does not alter the timing of puberty onset. A, Percentage of females experiencing VO from PND 22 to PND 31. B, Mean age at VO in females. C, Percentage of females experiencing FE from PND 30 to PND 44. D, Mean age at FE in females. E, Percentage of males experiencing preputial separation (PPS) from PND 22 to PND 34. F, Mean age at PPS in males (n = 6-9/genotype). G, H, Body weight throughout development and adulthood did not differ between WT and Kiss THKO females (G) and males (H; n = 4-6/genotype).
- Figure 3.
Basal gonadotropin levels and ovarian weights in females did not differ. LH (A) and FSH (B; n = 13/genotype) as well as ovarian weights (C; n = 6-8/genotype) did not differ between eight-week-old WT and Kiss THKO females.
- Figure 4.
Basal hormone levels and testes weights in eight-week-old WT and Kiss THKO male mice. LH (A) and FSH (B) did not differ between WT and Kiss THKO males (n = 13-15/genotype). T (C) levels were significantly higher in Kiss THKO males (*p < 0.05; n = 13-15/genotype). However, testes weights (D) did not significantly differ between WT and Kiss THKO males (n = 6-8/genotype).
- Figure 5.
The E2-mediated LH surge was not altered in Kiss THKO females. A, Percentage of eight-week-old females having an E2-mediated LH surge did not differ between WT and Kiss THKOs (n = 9-11/genotype). B, Neither genotype experienced an LH surge in the morning (n = 4-5/genotype), but both WT and Kiss THKOs had similar LH levels during the PM LH surge (n = 9-11/genotype). C, D, The number of corpora lutea did not differ between genotypes at eight weeks of age (n = 6/genotype). *, significant main effect of time, p < 0.05.
- Figure 6.
Kiss1 as well as cfos expression in Kiss1 and GnRH cells at the time of the E2-mediated LH surge did not differ between WT and Kiss THKO females. A, Photomicrograph of Kiss1 (silver grains) expression in the AVPV of adult WT and Kiss THKO female mice. B, Kiss1 levels in the AVPV/PeN of females did not differ between WT and Kiss THKOs. C, ISH photomicrograph of cfos (silver grains), a measure of gene activation, and Kiss1 (red fluorescence) colocalization (indicated by yellow arrows) in the AVPV/PeN of adult female mice in the PM (at the time of the LH surge). D, cfos expression in Kiss1 cells was low in the A.M. and elevated in the P.M. in both WT and Kiss THKOs. E, Photomicrograph of cfos (silver grains) and GnRH (red fluorescence) coexpression (indicated by yellow arrows) in the OVLT of adult female WT and Kiss THKO mice. F, cfos expression in GnRH cells was low in the A.M. and elevated in the P.M. (at the time of the LH surge) in both WT and Kiss THKOs. *, significant main effect of time, p < 0.05.
- Figure 7.
TH in Kiss1 cells is not required for normal fertility in females (A--D) or males (E--H). A, Percentage of WT and Kiss THKO females that had a litter during the 11-week fertility assessment. B, Mean latency (days) to first litter after pairing with the male. C, Mean total number of litters during the 11-week fertility study. D, Mean number of pups/litter (n = 5-8/genotype). E, Percentage of males siring at least one litter during the 11-week fertility assessment (n = 7/genotype). F, Mean latency (days) between the initial pairing with the female and when the first litter was born; measure only includes males that sired a litter. Mean total number of litters each male sired (G) and the mean number of pups/litter (H).
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