CREB Binding Protein (CBP) Activation Is Required for Luteinizing Hormone Beta Expression and Normal Fertility in Mice

GNRH induction of Lhb expression requires CBP phosphorylation at Ser436. (A) Total CBP protein levels from whole-cell lysates after LβT2 cells were transduced with either scrambled shRNA or CBP 5′ UTR Ad-shRNA, followed by transfection with pcDNA3.1 only, pcDNA expressing WT CBP (CBP-wt), or pcDNA expressing S436A mutant CBP (CBP-mut). (B) Relative Lhb transcript levels in LβT2 cells following transduction with adenovirus expressing CBP 5′UTR shRNA and transfection with pcDNA3.1, WT CBP, or S436A mutant CBP. Cells were treated with GNRH 4 h prior to the harvest of RNA. *, P < 0.01 versus values for WT untreated, pcDNA-transfected, and mutant untreated cells; #, P < 0.05. Bars represent means ± SEM.

CBP–Egr-1 interactions on the proximal Lhb promoter in response to GNRH. (A, B) CBP and Egr-1 occupancy of the Lhb promoter in LβT2 cells was detected by chromatin immunoprecipitation following GNRH treatment for the indicated periods of time. PCR (A) and Q-PCR (B) were performed using primers against both the proximal (−126 to −26) and the distal (−1189 to −893) Lhb promoter. *, P < 0.01 versus untreated cells. (C) LβT2 cells were transduced with Ad-shRNA against the 5′UTR of CBP, followed by transfection with the pFR-Luc reporter. Cells were also transfected as indicated with pcDNA only, WT CBP, or S436A CBP. Luciferase (Luc) activity was then measured after treatment with GNRH. *, P < 0.01 versus values for WT untreated cells and other conditions; #, P < 0.05. (D) Model of postulated CBP–Egr-1 interactions in the modified two-hybrid system. (E) CBP and Egr-1 occupancy of the Lhb promoter in response to GNRH was detected by chromatin immunoprecipitation in LβT2 cells 48 h after Egr-1 knockdown with Ad-shRNA. Real-time quantitative PCR was performed using primers against the proximal (−126 to −26) Lhb promoter. *, P < 0.01. (F) Immunoblot showing Egr-1 protein following transduction of LβT2 cells with scrambled or Egr-1 Ad-shRNA. Bars represent means ± SEM.

Assessment of fertility in S436A mice. Breeding pairs were established and observed for numbers of litters produced over time. (A) The number of pups per litter was observed over 60 days. Means without a common letter differ (P < 0.01). (B) Lengths of time to the first litter and between the first and second litters were measured for each breeding pair. Means without a common letter differ (P < 0.05). Bars represent means ± SEM. (C) Total number of litters and pups produced over 60 days for each breeding pair category. Each line represents values for one breeding pair, and each “×” represents a litter. M, male; F, female. There were 10 breeding pairs per group.

Assessment of the reproductive phenotype of S436A mice. (A) Vaginal cytology was performed at 24-h intervals by microscopic evaluation of stained vaginal smears, and the predominant cell type was recorded for each day. Data are shown for two representative animals from each group. (B) Hematoxylin- and eosin-stained histological sections of ovaries are shown from wild-type and S436A knock-in mice. Scale bars, 200 mm. (C) The proportion of time in diestrus versus estrus was recorded for each group. There were 7 to 8 mice per group. (D, E) Evening serum LH (D) and FSH (E) levels were measured in diestrus or proestrus for WT or S436A mice. There were 4 to 6 mice per group. *, P < 0.01; #, P < 0.05. Bars represent means ± SEM.

Assessment of phenotype in male mice. (A) Basal morning serum LH, serum FSH, and testosterone levels were measured in WT and S436A mice. There were 13 to 16 mice per group. No significant differences were detected. (B) Hematoxylin- and eosin-stained histological sections of testes are shown from wild-type and S436A knock-in mice. Scale bars, 200 mm. (C) Basal morning serum LH and FSH levels were measured in WT and S436A male mice 10 days following gonadectomy. There were 9 to 10 mice per group. (D) The fold change in serum LH was measured following subcutaneous administration of GNRH to WT and S436A male mice. GNRH stim, GNRH stimulation. There were 7 mice per group. *, P < 0.05 versus WT. Bars represent means ± SEM.