Quantitative Gonadotropin-Releasing Hormone: RESULTS(2)
To examine the quantitative amount of GnRH mRNA in the endometrial cells from individual subjects throughout the menstrual cycle, we coamplified the patient sample for 40 cycles in the presence of defined amounts of internal standard cDNA for GnRH. Figure 2 shows the standard curve obtained for GnRH mRNA by plotting the logarithmically transformed ratios of the densities of target cDNA to competitive cDNA against the log amount of initially added target cDNA in each PCR. The quantitative PCR of the different endometrial samples studied throughout the menstrual cycle showed a progressive increase in the mRNA levels from the early proliferative phase to the midluteal phase, which was statistically significant (p < 0.05) between these two phases (Fig. 4).
After the separation of the glandular epithelium from stromal cells, the presence of mRNA for GnRH and receptor in both endometrial compartments independently was demonstrated. Quantitative PCR of isolated glandular and stromal cells showed higher GnRH mRNA levels (p < 0.05) in the luteal phase than in the follicular phase in both compartments; however, the increase in mRNA levels was more pronounced in epithelial than in stromal cells (Fig. 5). buy asthma inhaler
Immunoreactive GnRH was present in all major endometrial compartments (luminal epithelium, glands, and stroma cells) throughout the entire menstrual cycle according to an avidin-biotin alkaline phosphatase technique. No staining was seen in cells of hematopoietic origin or vascular smooth muscle. The most intense immunoreactivity was noted during the luteal phase in all compartments as compared to the follicular phase (Fig. 6).
FIG. 4. Quantitative competitive PCRofGnRH mRNA from endometrial samples studied throughout the menstrual cycle. mRNA levels showed a progressive increase from the early proliferative phase to the midluteal phase. * p < 0.05 between these two phases.
FIG. 5. Quantitative, competitive PCR of isolated glandular and stromal cells. mRNA levels showed an increase in the luteal phase in both endometrial compartments; however, these increases were more evident in the epithelium than in the stroma. * and +, Bars with the same symbols showed significant differences at p < 0.05 and p < 0.01, respectively. Values are means ± SD.
FIG. 6. GnRH immunoreactivity in sections of human endometrium. a) Negative control; b and c) midluteal phase endometrium. GnRH immunoreactivity was highly expressed in the luminal epithelium (b) as well as in the stroma cells (c). X200.