HOXCW, HOXC11, HOXDW, and HOXD11 Expression Is Not Modulated by Sex Steroids in Ishikawa Cells
To determine if expression of HOXC10, HOXC11, HOXD10, and HOXD11 is regulated by sex steroids in epithelial cells, Ishikawa cell Hox gene RT-PCR transcript signal was measured after treatment with 17-p estradiol or progesterone. Cells were grown to 70%-80% confluence in steroid-free media and were transferred to serum-free media for 24 h before treating with physiologic concentrations of 17-p estradiol, progesterone, or both for 24 h. RNA was extracted and used for RT-PCR. HOXC10, HOXC11, HOXD10, and HOXD11 were expressed in Ishikawa cells (Fig. 5). Treatment with sex steroids did not significantly alter the signal representing the transcript level of any of these four genes. Each experiment was repeated five times. Densitometric analysis demonstrated the nonstatistically significant difference in response to 17-p estradiol or progesterone treatment in Ishikawa cells (data not shown).
HOXCW, HOXC11, HOXDW, and HOXD11 Are Expressed in Endometrial Stroma
To localize expression of HOXC10, HOXC11, HOXD10, and HOXD11, in situ hybridization was performed on proliferative and secretory phase endometrium. Figure 6 shows both bright-field and dark-field photomicrographs of representative sections from the proliferative phase. Each of these four HOX genes was expressed throughout the endometrium but not the myometrium. Expression appeared highest in the stroma, although signal above background was detected in the glandular epitheil-ium. The intensity of signal was consistent among multiple endometrial samples obtained from both the uterine fundus and the lower uterine segment. Hybridization to a control sense probe resulted in signal similar to background. Each hybridization was performed in duplicate on at least three tissue samples. Abundant expression was observed in proliferative phase endometrium confirming the results of the RT-PCR. Tissue obtained from the secretory phase also demonstrated stromal localization, though at much lower levels than in the proliferative phase (data not shown). These results similarly confirm the cyclic expression pattern identified by semiquantitative RT-PCR.
FIG. 5. HOXCIO, HOXC11, HOXDIO, and HOXD11 expression in Ish-ikawa cells in response to sex steroids. Cells were treated with 1 X 10~8 M 17^-estradiol (E), 1 X 10~6 M progesterone (P), or both hormones (E + P). There was no statistically significant difference between mRNA abundance of (A) HOXC10, (B) HOXC11, (C) HOXD10, and (D) HOXD11 among the treatment groups. Lane L: DNA ladder (100 bp). The experiment was repeated five times.
FIG. 6. HOXC10, HOXC11, HOXD10, and HOXD11 expression in endometrium during the menstrual cycle determined by in situ hybridization. Proliferative phase endometrium was obtained from normal-cycling women and hybridized to 33P labeled riboprobes specific to HOXC10, HOXC11, HOXD10, and HOXD11. All four HOX genes were expressed in the proliferative endometrium. Expression was highest in the endometrial stroma. Each hybridization was performed in duplicate on at least three tissue samples. Magnification X200.