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BOR - Papers in Press, published online ahead of print September 3, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.014969
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BIOLOGY OF REPRODUCTION 70, 39–45 (2004)
DOI: 10.1095/biolreprod.102.014969
© 2004 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

HOXC and HOXD Gene Expression in Human Endometrium: Lack of Redundancy with HOXA Paralogs1

G. Eda Akbas, and Hugh S. Taylor2

Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut 06520


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HOXA genes, essential regulators of differentiation in the embryo, are also essential for adult cyclic endometrial development and for endometrial receptivity. Mice deficient in Hoxa10 or Hoxa11 exhibit reduced fertility because of defects in implantation. We hypothesized that HOXC10, HOXC11, HOXD10, and HOXD11, paralogs of HOXA10 and HOXA11, might also be involved in endometrial development. Here, we showed that the expression of HOXC10, HOXC11, HOXD10, and HOXD11 was evident throughout the menstrual cycle in the endometrium by semiquantitative reverse transcription-polymerase chain reaction. In the secretory phase, expression of HOXC10, HOXC11, and HOXD11 decreased to 4% and HOXD10 decreased to 25% of the proliferative phase expression (P < 0.001, P < 0.001, P < 0.02, P < 0.01, respectively). In situ hybridization demonstrated expression of each of these HOX genes primarily in the stroma and confirmed the decreased expression in the secretory phase. HOXC10, HOXC11, HOXD10, and HOXD11 expression was not regulated by sex steroids in primary endometrial stromal cells or Ishikawa cells. The expression and regulation of HOXC and HOXD genes varies from that of HOXA10 and HOXA11. Whereas HOXA10 and HOXA11 are regulators of endometrial differentiation, HOXC and HOXD genes likely regulate endometrial proliferation. Paralogous HOX genes typically have a redundant function in development; a novel evolutionary divergence of paralogous Hox genes has resulted in HOXC and HOXD genes having distinct expression patterns, regulation, and likely also distinct functions from HOXA genes. A network of HOX genes may be involved in regulating multiple aspects of endometrial development, including both proliferation and differentiation.

female reproductive tract, implantation, menstrual cycle, Müllerian ducts, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Uterine endometrium is an extremely dynamic tissue, undergoing sequential developmental changes in preparation for implantation during each estrous cycle [1, 2]. The successful implantation of the blastocyst requires successful development of uterine endometrial receptivity [3–5]. In many ways, cyclic endometrial development in the adult can be considered analogous to embryonic development. Many of the genes traditionally thought of as regulators of embryonic development are also used to regulate endometrial development [6]. Hox genes, essential regulators of morphogenesis and tissue differentiation in the embryo, are also essential for endometrial development and for endometrial receptivity [7, 8].

Hox genes encode evolutionarily conserved transcription factors, which are important regulators of embryonic morphogenesis and differentiation [9, 10]. In mammals, 39 Hox genes reside in four separate chromosomal linkage groups, designated Hoxa, Hoxb, Hoxc, and Hoxd, each of which has parallel and overlapping expression domains [11]. Homologous members within the separate linkage groups are separated into 13 sets of paralogous genes, each having two to four members [12]. A distinguishing feature of the Hox genes is colinearity; their linear arrangement along the chromosome from 5' to 3' parallels their nested and segmental order of expression along the anterior-posterior body axis of the embryo during development [7, 12, 13]. Typically, paralogous HOX genes have similar or redundant functions and overlapping expression during embryonic development.

Two genes of the Hoxa cluster, Hoxa10 and Hoxa11, are expressed in localized areas of the paramesonephric duct destined to become the uterus or the lower uterine segment and cervix, respectively [11]. Hoxa10 and Hoxa11 gene expression is necessary for endometrial development, allowing uterine receptivity to implantation. Female Hoxa10 (-/-) or Hoxa11 (-/-) homozygous mutant mice have uterine factor infertility [14–17]. Although these mice ovulate normally, they are unable to support implantation. Embryos from Hoxa10 (-/-) mice successfully implant in pseudopregnant wild-type surrogates; however, wild-type embryos do not implant in Hoxa10- or Hoxa11-deficient uterus. In addition to regulating the embryonic development of the uterus [11], Hoxa10 and Hoxa11 have specific roles in endometrial development in the adult. Blocking maternal Hoxa10 expression in the adult uterus of wild-type mice with antisense blocks implantation, demonstrating the necessity of adult expression [18]. HOXA10 and HOXA11 are regulated by estrogen and progesterone in the adult human endometrium where their expression rises dramatically in the midsecretory phase, which is at the time of implantation, and remains elevated throughout the rest of the secretory phase [19, 20]. Human conditions associated with decreased implantation demonstrate diminished endometrial Hoxa10 and Hoxa11 expression [21–23].

The sixteen 5' Hox genes belonging to the paralogous groups 9–13 all show DNA sequence similarities to the Drosophila Abdominal-B (Abd-B) gene, which specifies the identity of the most posterior segments of the larval and adult fly. Among HOX genes, members of the HOXC and HOXD clusters, and in particular the Abd-B-related 5' genes of these linkage groups, are the least well characterized. In the developing genitalia, prominent expression of both Hoxc10 and Hoxc11 are observed in the posterior urogenital sinus, which gives rise to urethra and vagina [24]. Later, their expression is seen at high levels in paramesonephric duct and in the genital tubercle [24]. The expression patterns of Hoxc10 and Hoxc11 are similar; however, Hoxc10 is expressed more anteriorly in the paramesonephric duct, Hoxc11 is expressed only posterior to the pelvis, whereas Hoxc10 expression surrounds all the pelvic condensations and is also seen in the peritoneal and ventral epithelia. The posterior Hoxd genes are also expressed in female-developing genito-urinary tracts [25]. Genes of the Hoxd cluster have been shown to be expressed in the genital tubercle, the ovary, and the oviduct of the developing reproductive tract of mouse fetus [25]. Homozygous Hoxd10 mutant female mice are able to produce offspring [26]. Targeted disruption of both Hoxd9 and Hoxd10 does not alter fertility in female mice and does not result in abnormalities on uterine structure or position [27]. The HOXD10 gene has been shown to be expressed in the human uterus [28]. The expression of HOXD10 in tumors of the uterus, but not ovary or cervix, suggests that it too may also play a role in specifying human uterine identity [28]. Hoxd11 is expressed anteriorly from Hoxa11 or Hoxc11 [25]. No genito-urinary abnormality could be detected in Hoxd11 (-/-) female mice; loss-of-function females were fertile [29, 30]. The Hoxa11/Hoxd11/Hoxc11 triple mutant female mice are infertile and show many of the reproductive defects previously reported in Hoxa11 mutant mice [16, 31, 32]. Overlapping expression and redundant function among paralogous genes likely account for normal development when HOXC and HOXD genes are disrupted by homologous recombination.

The endometrium undergoes an ordered process of differentiation leading to receptivity to implantation. Coordinated expression of multiple HOX genes likely directs this development analogous to the way in which they direct embryonic development. The expression and regulation of HOXC10, HOXC11, HOXD10, and HOXD11, which are paralogs of HOXA10 and HOXA11, are not well characterized in the endometrium. Here, we investigated the expression and regulation of these genes in adult functional endometrial development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Collection

Endometrium was collected from 10 normal-cycling reproductive-age (20–35) women by endometrial biopsy with informed consent, under an approved Human Investigations Committee protocol. Tissue was immediately frozen in liquid nitrogen and stored at -80°C. Some of the tissue was fixed in formalin for histological examination or in paraformaldehyde for in situ hybridization. Menstrual-cycle dating was determined by menstrual history and confirmed by histological examination by using the criteria of Noyes et al [2].

Cell Culture

Endometrial samples were obtained from four different normal-cycling women in the proliferative phase. Endometrial epithelium and stromal cells were separated as described previously [33]. Briefly, endometrial tissue was digested by incubation of tissue minces in Hanks Balanced Salt Solution (HBSS) (Sigma, St. Louis, MO) that contained Hepes (25 mmol), penicillin (200 U/ml), streptomycin (200 mg/ml), collagenase (1 mg/ml, 15 U/mg), and deoxyribonuclease (0.1 mg/ml, 1500 U/mg) for 30 min at 37°C with agitation. The dispersed endometrial cells were separated by filtration through a wire sieve (73-µm diameter pore, Sigma). The stromal cells passed through the sieve into the filtrate, whereas the endometrial glands were retained by the sieve. The stromal cells were pelleted, washed, and suspended in phenol red-free charcoal stripped Hams F12/Dulbeccos minimal essential medium (1:1 vol/vol; Sigma) containing antibiotics and fetal bovine serum (FBS; 10% vol/vol; GIBCO BRL, Rockville, MD). Cells were plated in plastic flasks (75 cm2, Falcon, Franklin Lakes, NJ), maintained at 37°C in a humidified atmosphere (5% CO2 in air), and grown to confluence. The stromal cells were passed by trypsinization and plated in culture dishes (100-mm diameter) and were allowed to replicate to confluence. Immunocytochemical analysis of endometrial cells was conducted after the first passage. Factor VIII, cytokeratin, 3C10, and vimentin were used as markers of endothelial cells, epithelial cells, macrophages, and stromal cells, respectively. Ninety-seven percent of the cells were endometrial stromal cells. Epithelial cells and macrophages accounted for ~3% and 0.2% of the cells; endothelial cells were absent. The 70%–80% confluent monolayers were maintained in serum-free media for 24 h and subsequently treated with 17ß-estradiol (E2; 1 x 10-8 M; Sigma) or progesterone (1 x 10-6 M; Sigma) for 24 h. Steroids were dissolved in ethanol and diluted to a final concentration of less than 0.001%. Control cells were treated with an equal amount of ethanol.

Ishikawa cells, a well-differentiated endometrial adenocarcinoma cell line, were cultured in phenol red-free Eagles minimum essential medium containing 10% (v/v) charcoal-stripped FBS and supplemented with penicillin (100 µg/ml), glutamine (2 mM), and sodium pyruvate (1 mM). Estrogen and progesterone receptor status were verified by ELISA according to the manufacturer's instruction (Abbot Laboratories, Weisbaden, Germany). The 70%–80% confluent monolayers were maintained in serum-free media for 24 h and subsequently treated with 17ß-estradiol (E2; 1 x 10-8 M; Sigma) or progesterone (1 x 10-6 M; Sigma) for 24 h.

Semiquantitative Reverse Transcription-Polymerase Chain Reaction

QIAGEN RNAeasy kit (QIAGEN, Venlo, The Netherlands) was used to extract mRNA according to the manufacturer's instructions. Reverse transcription was carried out with 2 µg of sample in 20 µl of reaction mixture containing 10 mM each of dATP, dCTP, dGTP, and dTTP; 20 pmol oligo (dT); 40 u/µl of ribonuclease inhibitor; 10 u/µl of avian myeloblastosis virus-reverse transcriptase; and 5X AMV-RT buffer (42°C, 60 min; 95°C, 5 min: Eppendorf Mastercycler Gradient, Brinkmann, Westbury, NY). Primers that specifically amplified each of these genes were designed and tested; four pairs of primers were used for subsequent HOX amplification. The primers used for amplification of G3PDH were as described by Apostolakos et al [34]. The HOX primer sequences and product sizes are as follows: HOXC10, 5'-AAGACCTCAGACTCTCCTTCCAA-3' and 5'-GAGAACAGAATGCTGTGTGTGAG-3' (189 base pair [bp]); HOXC11, 5'-AATGTCTTGCTGCTCGGATTAG-3' and 5'-TAACACCAGGTTGAAGGTACAGAA-3' (225 bp); HOXD10, 5'-ATGTACATGCCACCACCTAGC-3' and 5'-TTGCTGTGTAACAGGTTGCTCTA-3' (192 bp); HOXD11, 5'-TGTACCTGCCGGGCTGCGCCTACTATGTGG-3' and 5'-GGCTGGACGTGCGGAGCCAGGTTGGAAGAGT-3' (130 bp); G3PDH, 5'-GGTCGGAGTCAACGGATTTGGTCG-3' and 5'-CTTCCGACGCCTGCTTCACCAC-3' (788 bp).

Amplification of cDNA by polymerase chain reaction (PCR) was performed using an annealing temperature and number of cycles optimized for each gene. For each PCR reaction, the number of cycles used was optimized so that the amplification process was carried out within the exponential (linear) range as demonstrated in Figure 1. To perform a semiquantitative analysis of samples, serial dilutions of cDNA were subjected to increasing PCR cycles in order to define the linear amplification range for each primer set. Four serial 10-fold dilutions of known amounts of HOXC10, HOXC11, HOXD10, HOXD11, or G3PDH cDNA were amplified in triplicate to construct standard curves for each primer pair. Twenty-five nanograms of each cDNA sample were then amplified at the optimal cycle number for each gene of interest. Coamplification of the transcript of interest with the internal, G3PDH, allowed comparison between samples. The PCR reaction was conducted in a total volume of 50 µl containing 10 x PCR buffer and 10 µM of each of 5' and 3' primers (95°C, 1 min; 65.7°C for HOXC10, 58.6°C for HOXC11, 65.7°C for HOXD10, and 66.2°C for HOXD11, 1 min; 72°C, 1.5 min). All the components for reverse transcription (RT)-PCR were purchased from Promega (Madison, WI) with the exception of the primers that were synthesized at the Yale University School of Medicine, Department of Pathology. 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. The gels presented in the figures are loaded for illustrative purposes and contain more PCR product than used for quantification.



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  		FIG. 1. Optimization of the RT-PCR by cycle number. Optimization of HOXC10 is shown as representative. Increasing cycle number provided an increased signal of both HOXC10 and G3PDH up to 30 cycles. MW, molecular weight markers

Statistical Analysis

The intensity of the bands was quantified by densitometry, and results were expressed as the ratio of intensity of HOXC10, HOXC11, HOXD10, and HOXD11 relative to G3PDH in all samples. Data were analyzed with the SigmaStat (Jandel Scientific, San Rafael, CA) software package. The values were expressed as mean ± SEM. Student t-test was used to compare results of HOX gene expression from endometrial biopsy samples representing proliferative and secretory phases. The differences between treatment groups in experiments with either stromal or Ishikawa cells were analyzed by one-way ANOVA. A statistically significant difference was defined as P < 0.05.

In Situ Hybridization

In situ hybridization was performed with antisense 33P-labeled riboprobes 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 Vectabond-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. Hybridization was performed in a humidified chamber for 16 h at 50°C. Slides were treated with RNase A at 37°C and were then washed for 16 h in 0.25 M NaCl, 0.01 M Tris-Cl (pH 7.5), 0.01 M sodium phosphate (pH 6.8), 5 mM EDTA, Ficoll 400 (0.02%), polyvinylpyrolidone (0.02%), BSA Fraction V (0.02%), and 50% formamide. Slides were dehydrated, dried, and dipped in K5D (Ilford Limited, Mobberley, Cheshire, United Kingdom) emulsion. Exposure was carried out at 4°C for 7–12 days, and slides were developed with D-19 (Eastman Kodak Co, Rochester, NY). Representative darkfield photomicrographs were taken at 20x on an Olympus microscope (Olympus Corp., Lake Success, NY). Slides subsequently were counterstained with hematoxylin and eosin, and corresponding bright-field photomicrographs were taken.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Endometrial Expression of HOXC10, HOXC11, HOXD10, 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.



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  		FIG. 2. HOXC10, HOXC11, HOXD10, and HOXD11 transcript abundance in endometrium during the menstrual cycle determined by semiquantitative RT-PCR. A) HOXC10 (189 bp), (B) HOXC11 (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)



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  		FIG. 3. Densitometric analysis of HOXC10, HOXC11, HOXD10, and HOXD11 signal in 10 samples normalized to G3PDH. In the secretory phase, expression of (A) HOXC10, (B) HOXC11, and (D) HOXD11 decreased to 4%, and (C) HOXD10 decreased to 25% of the proliferative phase expression (P < 0.001, P < 0.001, P < 0.02, P < 0.01, respectively). Values represent the mean ± SEM (n = 10). Each experiment was repeated three times

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-ß 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). However, treatment with sex steroids did not significantly alter the level of transcript of any of these four genes (Fig. 3). HOXA10 transcripts are increased with sex-steroid treatment as previously described and shown as a positive control [19]. Each experiment was repeated five times. Average densitometry readings were used to quantify and normalize the results; 17-ß estradiol, progesterone, or both combined did not significantly alter HOXC10, HOXC11, HOXD10, and HOXD11 expression in human stromal cells (data not shown).



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  		FIG. 4. HOXC10, HOXC11, HOXD10, and HOXD11 expression in stromal cells in response to sex steroids as demonstrated by RT-PCR. Cells that were demonstrated to be estrogen- and progesterone receptor-positive expressed (A) HOXC10, (B) HOXC11, (C) HOXD10, and (D) HOXD11. Treatment with 1 x 10-8 M 17ß-estradiol (E), 1 x 10-6 M progesterone (P), or both hormones (E + P) resulted in no statistically significant difference in HOXC10, HOXC11, HOXD10, and HOXD11 signal in between treatment groups. Lane L: DNA ladder (100 bp). The response of HOXA10 to E + P is shown as a control. Each experiment was repeated five times

HOXC10, HOXC11, HOXD10, 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-ß 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-ß 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-ß estradiol or progesterone treatment in Ishikawa cells (data not shown).



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  		FIG. 5. HOXC10, HOXC11, HOXD10, and HOXD11 expression in Ishikawa cells in response to sex steroids. Cells were treated with 1 x 10-8 M 17ß-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

HOXC10, HOXC11, HOXD10, 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 epitheilium. 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.



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  		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


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The endometrium undergoes cyclic developmental changes and an ordered process of differentiation in response to circulating sex steroids leading to receptivity to implantation [1]. 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 Müllerian system, and their expression persists in the adult female reproductive tract [11]. In development, both Hoxc10 and Hoxc11 are prominently expressed in the posterior urogenital sinus [24]. Later, high levels of expression can be detected in paramesonephric duct and in the genital tubercle [24]. The HOXD10 gene has been shown to be expressed in the human uterus [28], and Hoxd11 transcripts are expressed more anteriorly in the developing Müllerian tract [25]. Although HOXA10 and HOXA11 clearly have a role in cyclic functional human endometrial development resulting in embryonic receptivity, the role of their paralogs has not been defined.

The expression pattern of HOXC10, HOXC11, HOXD10, and HOXD11 in the human endometrium through the menstrual cycle differs from that of HOXA10 and HOXA11, both of which rise dramatically in the midluteal phase. Whereas HOXA10 and HOXA11 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.

Although HOX gene expression has been classically associated with differentiation, recent studies have suggested a role in proliferation [35]. At the start of cell division, proteins must be assembled onto replication origins to establish competence for initiation of DNA synthesis. The selection and activation of these replication origins is the key process in controlling chromosome replication during cell proliferation [36]. HOXC10 protein binds a 74-bp sequence within the human DNA replication origin associated with the Lamin B2 gene in Cos7 cells [35]. Additionally, HOXC10 is highly expressed in two exponentially growing cell lines, namely HeLa S3 cells [37] and monocytic U937 cells. 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. HOXC10 is degraded early in mitosis by the cell-cycle regulatory enzyme, anaphase-promoting complex, further indicating a role in cell-cycle progression and proliferation [38]. Together, these results indicate that HOXC10 is expressed not only during differentiation, as expected for a homeoprotein, but also in response to proliferative stimuli. HOXC10 may have a similar role in the early development of endometrium and endometrial proliferation. This hypothesis is supported by the high levels of HOXC10 expression that we detected in the proliferative phase as compared with the secretory phase in the endometrium. Similarly, HOXC11, HOXD10, and HOXD11 may be involved in proliferation rather than differentiation.

HOXC10, HOXC11, HOXD10, and HOXD11 expression is noted in the proliferative phase of the menstrual cycle, when estrogen is the predominant steroid hormone affecting the uterus. HOXC10, HOXC11, HOXD10, and HOXD11 mRNA levels dramatically decrease in the secretory phase at the time when progesterone levels rise rapidly. We had previously shown that HOXA10 and HOXA11 expression is rapidly induced in response to estrogen and progesterone in both stromal and Ishikawa cells [19, 20]. However, HOXC10, HOXC11, HOXD10, and HOXD11 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 [7] and can act as transcriptional repressors of other homeobox genes [39]. HOXC and HOXD genes may be regulated by HOXA genes or other transcriptional regulators.

Hox genes of any given paralogs are thought to function redundantly. Abd-B-related mammalian HOXC and HOXD genes may regulate identical or functionally equivalent downstream targets that may be involved in the regulation of cell proliferation during proliferative phase in the endometrium. The evidence showing overlapping function of Hox genes in mammals is particularly strong for members of any paralogous group. For most paralogous groups the encoded homeodomains are nearly identical, differing by zero to six amino acids, and Hox proteins have similar in vitro DNA target-binding specificities. 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 [30–32, 40–46]. Loss of function of one member of the paralogous group results in hypomorphic 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 [27].

Here, we demonstrate that HOXC and HOXD genes likely are not redundant to HOXA genes in the endometrium. HOXC10, HOXC11, HOXD10, and HOXD11 are expressed at high levels in the proliferative phase, and their expression decreases in the secretory phase. HOXA10 and HOXA11 are both up-regulated by sex steroids in the midluteal phase. Our data suggest a lack of direct sex-steroid effect on HOXC10, HOXC11, HOXD10, and HOXD11 mRNA abundance in the endometrium. Unlike HOX redundancy in embryonic development, this differential adult expression suggests distinct functions. This novel lack of redundancy in the adult uterus suggests that in eutherian species Hox paralogs function has diverged with the evolution of distinct methods of reproduction. The duplication of Hox clusters may have allowed for evolution of adult reproductive tract plasticity and novel functions such as an estrous cycle.


   FOOTNOTES
 
1 This work was supported by NIH grant HD 36887. Back

2 Correspondence: Hugh S. Taylor, Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar St., P.O. Box 208063, New Haven, CT 06520-8063. FAX: 203 785 7134; hugh.taylor{at}yale.edu Back

Received: 23 December 2003.

First decision: 7 January 2003.

Accepted: 14 August 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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