Endometrial Expression of HOXCIO, HOXC11, HOXDIO, and HOXD11 Is Menstrual Cycle-Stage Dependent
To determine the transcript level of HOXC10, HOXC11, HOXD10, and HOXD11 in the cyclic development of the endometrium, the menstrual cycle stage-specific RT-PCR signal was characterized. Human endometrium was collected from normal-cycling women, and mRNA was extracted. The 10 specimens were separated into two approximately equal groups corresponding to proliferative (early and late) stage and to secretory (early, mid, and late) stage endometrium.
Four pairs of primers that were designed to specifically amplify a segment of each of these genes, and an annealing temperature and number of cycles optimized for each gene, were used in RT-PCR as demonstrated in Figure 1. The identity of PCR products was confirmed by sequencing. Representative results of HOXC10- (189 bp), HOXC11- (225 bp), HOXD10- (192 bp), HOXD11- (130 bp), and G3PDH- (788 bp) specific RT-PCR products are shown in Figure 2. Signal was evident throughout the menstrual cycle but decreased dramatically in the midsecretory phase. G3PDH was used as a control. Densitometric analysis was performed on each sample, and Figure 3 displays the average abundance of HOXC10, HOXC11, HOXD10, and HOXD11 during the proliferative stage of the menstrual cycle normalized to G3PDH. In the secretory phase, the signal representing expression of HOXC10, HOXC11, and HOXD11 decreased to 4% of proliferative phase expression (P < 0.001, P < 0.001, P < 0.02, respectively), and HOXD10 decreased to 25% of the proliferative phase expression (P < 0.01). The signal representing HOXC10, HOXC11, HOXD10, and HOXD11 expression varied during the menstrual cycle and markedly decreased at the secretory phase.
FIG. 1. Optimization of the RT-PCR by cycle number. Optimization of HOXCIO is shown as representative. Increasing cycle number provided an increased signal of both HOXC10 and G3PDH up to 30 cycles. MW, molecular weight markers.
FIG. 2. HOXC10, HOXC11, HOXD10, and HOXD11 transcript abundance in endometrium during the menstrual cycle determined by semi-quantitative RT-PCR. A) HOXC10 (189 bp), (B) HOXC1 1 (225 bp), (C) HOXD10 (192 bp), (D) HOXD11 (130 bp), and G3PDH (788 bp) are expressed in early proliferative (EP) phase, late proliferative (LP) phase, early secretory (ES) phase, mid secretory (MS) phase, and late secretory (LS) phase. A significant decrease in signal occurs in the ES phase and persists into the LS phase. Lane L: DNA ladder (100 bp).