Figure 1
Characterization of LH/CGR transcript variants and protein in the monkey CL throughout the luteal phase. (A) Circulating mean serum P4 levels and LH/CGR mRNA expression determined during different stages of luteal phase. Individual bars represent mean ± SEM values (n = 3 animals/stage). For comparison among different stages of luteal phase, one-way ANOVA analysis is performed and bars with different letters are significantly (p < 0.05) different. (B) Northern blot analysis of LH/CGR mRNA in CL obtained from different stages of the luteal phase and during regression. Four transcripts of LH/CGR mRNA migrating with apparent sizes of 1.0, 4.0, 7.5 and 8.0 kb are indicated by arrows. The 28S and 18S ribosomal RNA corresponding to the sizes of 5 kb and 1.86 kb, respectively are also shown. A comparative analysis of LH/CGR mRNA expression between mid and late stage of luteal phase indicated presence of all four transcripts in the late stage CL. The blots were reprobed with α 32P labeled L-19 cDNA probe to confirm for equal loading of total RNA. The data shown is for one of three northern blots (n = 3 CL/stage). (C) Semi-quantitative RT-PCR analysis of the LH/CGR mRNA in CL tissues collected during different stages of the luteal phase. In the upper panel, representative gel pictures of the products obtained after employing two sets of primer pairs designed spanning exon 5-11 and exon 7-11 regions, respectively. In the lower panel, representative gel pictures of the products obtained employing two sets of primer pairs designed spanning exon 11 and exon 9 regions, respectively. The positions of amplified products are indicated by arrows. (D) Levels of LH/CGR protein in the monkey CL throughout the luteal phase. The immunoblot probed with anti-β-actin antibody was utilized to confirm that the equal amount of protein was loaded in each lane. Densitometric analysis (mean ± SEM) of immunoblots was performed and the protein level in the mid luteal phase was set as one fold and protein levels in other stages relative to the mid luteal phase are indicated below the respective bands. (E) Circulating P4 levels before and 2 h after rhLH (20 IU/kg BW) treatment on day 1 of menses. Each bar represents mean ± SEM values of six independent experiments. Different letters on bars indicate statistical significance (p < 0.05).