HOXC and HOXD Gene Expression in Human Endometrium: DISCUSSION(5)

Loss of function of two or more adjacent, paralogous, or similarly expressed Hox genes has repeatedly demonstrated exacerbations of phenotype compared with the single mutants, often unmasking phenotypes not evident after single mutation of any of the genes involved. Loss of function of one member of the paralogous group results in hypo-morphic phenotypes. These observations suggest functional redundancy among the Hox genes, such that inactivation of one gene can be compensated by the activity of other Hox genes, which share sequences or have similar domains of expression.

HOXC and HOXD Gene Expression in Human Endometrium: DISCUSSION(4)

DISCUSSION(4)

H0XC10, H0XC11, H0XD10, and H0XD11 mRNA levels dramatically decrease in the secretory phase at the time when progesterone levels rise rapidly. We had previously shown that H0XA10 and H0XA11 expression is rapidly induced in response to estrogen and progesterone in both stromal and Ishikawa cells. However, H0XC10, H0XC11, H0XD10, and H0XD11 expression was not altered by estrogen or progesterone in either primary stromal cells or Ishikawa endometrial adenocarcinoma cells. Hox genes typically cross-regulate each other’s activity and can act as transcriptional repressors of other homeobox genes. HOXC and HOXD genes may be regulated by HOXA genes or other transcriptional regulators.

HOXC and HOXD Gene Expression in Human Endometrium: DISCUSSION(3)

Similarly, Hoxc10 is expressed in exponentially growing mouse C2C12 myoblasts; however, Hoxc10 protein was undetectable both in quiescent myoblasts after serum starvation and in differentiated myotubes. H0XC10 is degraded early in mitosis by the cell-cycle regulatory enzyme, anaphase-promoting complex, further indicating a role in cell-cycle progression and proliferation.

HOXC and HOXD Gene Expression in Human Endometrium: DISCUSSION(2)

DISCUSSION(2)

The expression pattern of H0XC10, H0XC11, H0XD10, and H0XD11 in the human endometrium through the menstrual cycle differs from that of HOXA10 and H0XA11, both of which rise dramatically in the mid-luteal phase. Whereas H0XA10 and H0XA11 are regulators of endometrial receptivity, HOXC and HOXD genes may have a role in the early development of endometrium and endometrial proliferation rather than in differentiation and receptivity to embryonic implantation. A network of HOX genes may be involved in regulating multiple aspects of endometrial development, including both proliferation and differentiation.

HOXC and HOXD Gene Expression in Human Endometrium: DISCUSSION(1)

The endometrium undergoes cyclic developmental changes and an ordered process of differentiation in response to circulating sex steroids leading to receptivity to implantation. The molecular mechanisms that regulate the ordered proliferation, differentiation, and apoptosis during the normal menstrual cycle are poorly characterized. HOX genes are likely to have a role in the cyclic development of the endometrium in a way similar to that by which they direct embryonic development and the development of the reproductive tract. HOX A genes have been identified in the developing Mullerian system, and their expression persists in the adult female reproductive tract.

HOXC and HOXD Gene Expression in Human Endometrium: RESULTS(3)

 RESULTS(3)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).

HOXC and HOXD Gene Expression in Human Endometrium: RESULTS(2)

HOXC10, HOXC11, HOXD10, and HOXD11 Expression Is Not Modulated by Sex Steroids in Primary Human Endometrial Stromal Cells

To test whether HOXC10, HOXC11, HOXD10, and HOXD11 expression is regulated by sex steroids, the signal corresponding to each HOX transcript was measured in primary human stromal cells after treatment with estrogen or progesterone. Endometrial samples from proliferative phase of the menstrual cycle were used as a source of primary cultures of stromal cells. Cells were grown to confluence in charcoal-stripped, phenol red-free media and serum-starved for 24 h before a 24-h treatment with physiologic concentrations of 17-p estradiol, progesterone, or both. RNA was extracted and used for RT-PCR. HOXC10, HOXC11, HOXD10, and HOXD11 mRNA was idetified in the endometrial stromal cells (Fig. 4).

HOXC and HOXD Gene Expression in Human Endometrium: RESULTS(1)

RESULTS(1)

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.

HOXC and HOXD Gene Expression in Human Endometrium: MATERIALS AND METHODS(6)

In Situ Hybridization

In situ hybridization was performed with antisense 33P-labeled ribo-probes specific to HOXC10, HOXC11, HOXD10, and HOXD11. Probes are a gift of E. Boncinelli and have been previously characterized. Endometrium was fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose, and then embedded in OCT compound (Miles Laboratories, Elkhart, IN). Ten-micrometer frozen sections were obtained and mounted on Vec-tabond-coated slides (Vector Laboratories, Inc., Burlingame, CA). Before use, sections were treated with 0.2 M HCl, Pronase (0.16 mg/ml), and 0.026 M acetic anhydride and were then dehydrated. Tissue sections were hybridized overnight with 3 X 106 cpm of each probe in 0.25 M NaCl, 0.01 M Tris-HCl (pH 7.5), 0.01 M NaPO4 (pH 6.8), 5 mM EDTA, Ficoll 400 (0.02%), polyvinylpyrolidone (0.02%), BSA Fraction V (0.02%), 50% formamide, 12.5% dextran sulfate, yeast tRNA (1.25 mg/ml), and 10 mM DTT.

HOXC and HOXD Gene Expression in Human Endometrium: MATERIALS AND METHODS(5)

METHODS(5)

Each reaction was repeated five times. The PCR products were separated on 2.5% agarose-TAE (40 mM Tris-acetate, 1 mM EDTA) gels containing ethidium bromide (10 mg/ml) and visualized by UV light. Representative RT-PCR products were excised from agarose gels and confirmed by DNA sequencing. Expression of HOXC10, HOXC11, HOXD10, HOXD11, and G3PDH were assessed on unsaturated gels by densitometric quantification by using laser densitometry, and HOX values were normalized to G3PDH. RT-PCR values are presented as a ratio of the specified gene’s signal in the selected linear amplification cycle divided by the G3PDH signal.