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Endocrinology, doi:10.1210/en.2006-0231
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Endocrinology Vol. 147, No. 9 4079-4092
Copyright © 2006 by The Endocrine Society

Indian Hedgehog, But Not Histidine Decarboxylase or Amphiregulin, Is a Progesterone-Regulated Uterine Gene in Hamsters

Atanu Khatua, Xiaohong Wang, Tianbing Ding, Qian Zhang, Jeff Reese, Francesco J. DeMayo and Bibhash C. Paria

Divisions of Reproductive and Developmental Biology (A.K., X.W., T.D., Q.Z., B.C.P.) and Neonatology (J.R.), Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2678; and Department of Molecular and Cellular Biology (F.J.D.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Bibhash C. Paria, Division of Reproductive and Developmental Biology, D4124 Medical Center North, 1161 21st Avenue South, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2678. E-mail: bc.paria{at}vanderbilt.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Implantation occurs only in the progesterone (P4)-primed uterus in the majority of species, but little effort has been given to identify P4-mediated molecules in these species. Using hamsters as a model for P4-dependent implantation and three well-known uterine receptivity-associated P4-regulated genes, Indian hedgehog (Ihh), histidine decarboxylase (Hdc), and amphiregulin (Areg), in mice that require ovarian estrogen for uterine receptivity and implantation, our strategy aimed to determine whether P4 regulates uterine expression of these genes in hamsters and whether the event- and cell-specific uterine expression patterns of these genes during the periimplantation period in hamsters follow similarly with their patterns in mice. We report here that P4-mediated Ihh signaling is important for uterine receptivity and implantation in hamsters because uterine epithelial Ihh expression was regulated by P4 and its expression patterns during the periimplantation period of hamsters closely follow its pattern in mice. In contrast, we noted no hormonal regulation of Hdc and Areg in the hamster uterus. However, this did not diminish their importance in hamsters because their expression patterns and functions are event and cell specific during the periimplantation period: whereas Hdc was expressed exclusively in d 4 uterine glands and regulated by the blastocyst, Areg was expressed on the decidual area adjacent to the embryo from d 5 onward and involved in stromal cell proliferation. We conclude that similarities and dissimilarities exist in uterine expression pattern of implantation-related genes, including hormonal regulation and their event-specific importance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MOLECULES REQUIRED FOR specific reproductive events may vary from species to species. In this respect, the hormonal requirement for uterine receptivity and implantation is not an exception. The work of Krehbiel (1) first showed a need for estrogen (E) in initiation of implantation in pregnant lactating rats. This observation was later confirmed and extended to mice showing an absolute E dependency in initiation of implantation in the progesterone (P4)-primed uterus of these two species (2, 3, 4). However, several studies showed that daily injection of P4 alone initiates implantation in ovariectomized and hypophysectomized hamsters (5, 6, 7). Orsini and Meyer (7) even demonstrated that daily injection with as little as 62 µg P4 resulted in nidation in 50% of hamsters that were ovariectomized on d 2 of pregnancy. These results indicated that in hamsters, the blastocyst implantation process is P4 dependent and the uterus is sensitive to small amounts of P4. Similarly, the P4-dependent implantation process was also observed in rabbits, pigs, guinea pigs, and monkeys (8, 9, 10, 11, 12, 13, 14). Moreover, studies demonstrated that luteal E may not be a requirement for implantation in humans (15). Hence, it remains to be seen what makes the uterus receptive solely in response to P4 in certain species but not in others. A clue in this regard can be obtained by simple assessment and comparison of the expression patterns of known P4-regulated uterine genes between the species in which implantation is either E or P4 dependent.

Among several uterine genes, Indian hedgehog (Ihh), histidine decarboxylase (Hdc), and amphiregulin (Areg) are well-recognized P4-regulated genes in the ovariectomized mouse uterus (16, 17, 18, 19). Ihh is a member of the mammalian hedgehog family that is mainly involved in the regulation of bone and pattern formations (20). Hdc is a rate-limiting enzyme in the formation of histamine that is involved in inflammatory reactions (reviewed in Ref. 16). Areg is a member of the epidermal growth factor family and is mainly involved in cell proliferation and differentiation (21). The d 4 pregnant uterus of mice showed maximum expression of these three genes, and hence, their expression patterns were considered to be closely related to the time of P4 dominance in uterine preparation for implantation, suggesting their possible regulation by P4 and involvement in uterine receptivity and implantation in this species (16, 17, 18, 19). However, the hormonal regulation and expression patterns of these genes during early pregnancy in the uterus have not been studied in detail in species in which uterine receptivity and implantation is only P4 dependent.

In this study, using the hamster as an animal model for P4-dependent implantation, we first investigated the influence of steroid hormones in the regulation of uterine expression of Ihh, Hdc, and Areg genes in ovariectomized hamsters. In addition, we also studied the expression patterns of these genes during early pregnancy in relation to uterine receptivity, initiation of implantation, and the decidualization process. Finally, we determined whether any major differences exist in the hormonal regulation and expression of the Ihh, Hdc, and Areg genes in the early pregnant hamster uterus, compared with the data reported in the mouse.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and treatments
Adult virgin golden hamsters (Mesocricetus auratus) and CD1 mice were purchased from Charles River Laboratory (Wilmington, MA) and kept under controlled temperature and lighting (lights on from 0600–1800 h). All animals were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory animals, and procedures were approved by our Institutional Animal Care and Use Committee. The 4-d estrous cycle of hamsters was monitored by the presence of characteristic vaginal discharge on the morning of the day of ovulation, which was designated d 1 of the estrous cycle; d 4 corresponds to proestrus (22, 23, 24). Hamsters exhibiting at least three consecutive regular 4-d estrous cycles were used in this study.

To determine the effects of P4 and E on uterine gene expression, hamsters and mice were ovariectomized, regardless of their stage of the estrous cycle, and rested 15 d to eliminate circulating ovarian steroids. Control animals were injected with vehicle (sesame seed oil; 0.1 ml/animal). Experimental animals received a single injection of P4 (2 mg per 0.1 ml/mouse; 2 mg per 0.1 ml/hamster), estradiol-17ß (E2; 100 ng per 0.1 ml/mouse; 1000 ng per 0.1 ml/hamster), or a combination of the same doses of P4 plus E2 or one dose of P4 for 2 d and a combination of the same doses of P4 and E2 on the third day. Control animals were killed at a single time point 24 h after an injection of vehicle sesame seed oil. Experimental animals with single injection of P4 or E2 were killed at 3, 6, 12, and 24 h after steroid injection. Uteri were removed, freed from adjacent fat and mesentery, sliced into small pieces, and immediately snap frozen in cold Friendly Freeze’it (Curtin Matheson Scientific, Houston, TX) and stored at –70 C until processed for RNA isolation or cutting sections for Northern blot or in situ hybridization, respectively. Experimental animals that received a combination of P4 and E2 were killed at 6, 12, and 24 h after the last injection. These uterine tissues were collected only for in situ hybridization. The dose of P4 was selected on the basis of its ability to maintain pregnancy in ovariectomized or hypophysectomized hamsters (22, 23, 24). The dose of E2 was selected based on its ability to stimulate heparin-binding epidermal growth factor-like growth factor (HB-EGF) gene expression in ovariectomized uteri of hamsters (22). All steroids were dissolved in sesame oil and injected sc. Unless otherwise mentioned, all reagents were purchased from either Sigma-Aldrich Co. (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA).

For the collection of uterine tissues during early pregnancy, pregnant hamsters were prepared by caging a single female with two fertile males on the evening of the proestrus. Vaginal smears were examined for the presence of spermatozoa on the following morning, which was designated as d 1 of pregnancy (22, 23, 24). Pregnant mice were prepared by caging a single fertile male with three females regardless of their stage of the estrous cycle. The female mice were checked for the presence of a vaginal plug the next morning. The finding of a vaginal plug was designated as d 1 of pregnancy in mice (16, 22, 23, 24). Hamsters on d 1–3 of pregnancy were killed at 0830–0900 h. Their whole uteri were removed and cut into small pieces after confirmation of pregnancy by flushing and recovering appropriate stages of embryos from oviducts. Whereas whole uteri were collected on d 4 morning (0900 h), implantation sites were collected from hamsters and mice on the morning (0900 h) of d 5 after an iv injection of Chicago Blue B dye solution (0.25 ml of 1% dye in saline per hamster; 0.10 ml of 1% dye in saline per mouse) (22, 23, 24). Implantation sites on d 5 were visualized as intermittent blue bands along the horns. On d 6–8, implantation sites were distinct and identified visually without blue dye injection (22, 23, 24). Uterine tissues were immediately frozen and stored at –70 C for in situ hybridization studies.

To determine the influence of the embryo on uterine gene expression, one oviduct of a pregnant hamster was cut at the uterotubal junction on d 2 of pregnancy to prevent the entry of embryos inside that uterine horn. The contralateral uterine horn and the oviduct of this animal remained intact for normal embryo entry into the uterus. These animals were killed on d 4 (0900 h), and uterine horns with or without embryos were cut into small pieces and collected separately. Uterine Ihh and Hdc, expression patterns were compared between uterine tissues with or without blastocysts by in situ hybridization.

Implantation occurs without delay in hamsters ovariectomized or hypophysectomized on d 2 of pregnancy and given P4 daily (22, 23, 24). This suggests that implantation in hamsters is P4 dependent. Thus, it is possible that implantation occurred with correct expression of implantation-specific genes because of regulation of these genes by either P4 or blastocysts. To address this issue, a group of pregnant hamsters was ovariectomized on d 2 (0900 h) and given a sc injection of P4 (1 mg in 0.1 ml sesame seed oil per hamster) on d 2–5 to maintain pregnancy. A short segment of suture was placed inside the lumen of one uterine horn on d 3 of pregnancy for the purpose of disturbing normal embryo-induced implantation and inducing deciduoma experimentally in this horn. The contralateral horn of this animal was kept intact for normal embryo-induced implantation and decidualization. Animals were killed on d 6 after an iv blue dye injection (22, 23), and the embryo-induced decidual and suture-induced deciduomal tissues were collected separately. Expression of the Areg gene was compared between the decidual and deciduomal tissues by in situ hybridization.

Immunohistochemical localization of hamster uterine Ihh, Areg, and Hdc proteins was not performed due to unavailability of antibodies against these hamster antigens and failure to recognize hamster antigens by several of these commercially available antibodies raised against either the mouse or the human antigens.

Cloning of the hamster Hdc, Areg, and Ihh partial cDNAs
RT-PCR was used to generate the hamster-specific Hdc, Areg, and Ihh partial cDNAs. The sequences of oligonucleotides used to generate cDNAs are as follows: Hdc (GenBank accession no. NM 008230, spanning nucleotides 351–821), forward 5'-CGC CTA CTA TCC TGC TCT TAC C-3', reverse 5'-CCC TGT TGC TTG TCT TCC TC-3', size 471 bp; Areg (GenBank accession no. NM 017123, spanning nucleotides 167–690), forward 5'-GCG GAA CCA ATG AGA ACT CC-3', reverse 5'-CAC CGT TCG CCA AAG TAA TC, size 524 bp; and Ihh (GenBank accession no. NM 010544, spanning nucleotides 991-1220), forward 5'-CGT GCA TTG CTC TGT CAA GT-3' and reverse 5'-CTC GAT GAC CTG GAA AGC TC-3', size 229 bp. Total RNAs (1 µg) from d 4 and 5 of pregnant hamster uteri were reversed transcribed with oligo-dT at 42 C for 50 min using the SuperScript first-strand synthesis system (Invitrogen Corp., Carlsbad, CA). PCR was carried out with an initial denaturation of 95 C for 10 min followed by 40 cycles consisting of denaturation at 94 C for 1 min, annealing at 64 C for 30 sec, and elongation at 72 C for 30 sec. This was followed by a final extension at 72 C for 8 min. RT-PCR-generated uterine Hdc, Areg, and Ihh products were subcloned into a pCR-II-TOPO cloning vector (3.9 kb) using a TOPO TA cloning kit (version K2; Invitrogen) (23), and nucleotide sequences of these clones were determined on both strands to verify the clone’s identity. The GenBank accession number for the hamster Ihh, Hdc, and Areg are DQ319998, AF345330, and AY135447, respectively. Nucleotide sequences of these partial cDNA clones showed more than 90% sequence similarity with that of GenBank nucleotide database for rats, mice, and humans.

RNA probe preparation
For Northern blot and in situ hybridizations, plasmids bearing hamster cDNA were extracted, purified, and linearized to generate antisense and sense riboprobes, which were transcribed using appropriate RNA polymerases (Hdc: T7/HindIII for sense and SP6/XhoI for antisense; Areg: T7/BamH1 for sense and SP6/Not1 for antisense; Ihh: T7/BamH1 for sense and SP6/Not1 for antisense) and labeled with 32P or 35S, respectively (23). A partial clone of rpL7 cDNA was also used as a template for the synthesis of 32P-labeled antisense cRNA probe. All labeled sense and antisense cRNA probes used for hybridizations had specific activities of approximately 2 x 109 dpm/µl.

Isolation of RNA
Total RNA was extracted from whole uteri by a modified guanidine thiocyanate procedure (16, 23). Uteri pooled from three animals were homogenized in 10 ml of 5 M guanidine thiocyanate, 25 mM EDTA, 50 mM Tris (pH 7.4), and 8% (vol/vol) ß-mercaptoethanol, and nucleic acids were precipitated with 3 ml cold ethanol. The precipitate was dissolved in 6 M guanidine hydrochloride, 25 mM EDTA, and 10 mM ß-mercaptoethanol, and RNA was precipitated for 3 h at –20 C after the addition of 0.5 vol of cold ethanol and 0.05 vol of 1 M acetic acid. This procedure was repeated twice, and the final pellet was dissolved in alkaline buffer [75 mM NaCl, 25 mM EDTA, and 0.5% (wt/vol) sodium dodecyl sulfate], phenol chloroform extracted, brought to 0.3 M ammonium acetate, and precipitated with ethanol at –20 C for 3 h. The precipitate was rinsed in 70% (vol/vol) ethanol and dissolved in water and RNA was quantitated by measuring the absorbance at 260 nm.

Northern blot hybridization
Total RNA (6 µg) was separated by formaldehyde-agarose gel electrophoresis, Northern blotted to nitrocellulose membranes, and cross-linked to the membrane by UV irradiation. Northern blots were prehybridized, hybridized, and washed as previously described (16, 22). The blots were sequentially hybridized with 32P-labeled Hdc, Areg, Ihh, and Rpl7 probes, and the hybrids were detected by autoradiography. Bands resulting from radioactive hybrids were analyzed using National Institutes of Health ImageJ 1.36b to determine their relative intensities (OD). For normalization, the intensity of each band was divided by the value of the corresponding Rpl7 band intensity. Each Northern blot hybridization experiment was performed three times to ensure reproducibility of results.

In situ hybridization
The protocol was followed as described by Paria and co-workers (16, 23). Briefly, uterine cryosections (10 µm) were mounted onto poly-L-lysine-coated slides and fixed in cold 4% paraformaldehyde solution in PBS for 15 min on ice. After prehybridization, sections were hybridized to 35S-labeled antisense probes at 45 C for 4 h in 50% formamide hybridization buffer. Parallel sections were hybridized with 35S-labeled sense probes as negative control. After hybridization and washing, sections were incubated with ribonuclease (RNase) A (20 µg/ml) at 37 C for 20 min. RNase A-resistant hybrids were detected by autoradiography using Kodak NTB-2 liquid emulsion (Eastman Kodak Co., Rochester, NY). The slides were poststained with hematoxylin and eosin. Each in situ hybridization experiment was performed three times to ensure reproducibility of results.

Primary culture of uterine stromal cells
Primary culture of uterine stromal cells was performed according to the protocol as previously described (25) with minor modifications. In brief, uterine horns were collected from d 4 pregnant hamsters, cleaned of adherent fat tissues, slit longitudinally, and cut into small pieces. Uterine pieces were placed in a sterile petri dish and washed thoroughly with Hanks’ balanced salt solution (HBSS; Invitrogen) without Ca+2/Mg+2 and phenol red but containing 100 U/ml penicillin (Invitrogen); 100 µg/ml streptomycin (Invitrogen), and 2.5 µg/ml amphotericin B (Sigma). Tissues were then placed in fresh HBSS containing 6 mg/ml dispase (Invitrogen) and 25 mg/ml pancreatin and incubated in sequence for 1 h at 4 C, 1 h at room temperature, and 10 min at 37 C. After these digestion steps, tissues were diluted in HBSS (17 ml) containing 10% (wt/vol) charcoal-stripped fetal bovine serum (FBS) and mixed thoroughly to dislodge the sheet of luminal epithelial cells by pipetting up and down several times using 25 ml pipette. After a brief centrifugation, the supernatants containing epithelial cells were discarded. The remaining tissues were washed twice in fresh HBSS and incubated for subsequent digestion in a fresh medium (3 ml) containing 0.5 mg/ml collagenase type 1 (Invitrogen) at 37 C for 30 min. At the end of digestion, tissues were diluted in HBSS (17 ml) with 10% charcoal-stripped FBS and mixed thoroughly using a 25-ml pipette. The digested cells (primarily containing stromal cells) were then passed through a 70-µm nylon filter to eliminate clumps of epithelial cells and centrifuged. The precipitates were washed twice with HBSS before the initiation of the primary culture. Cells were seeded at 5 x 105 cells per 60 x 15 mm polystyrene tissue culture dish or, as indicated otherwise in the text, containing phenol red-free DMEM and Ham’s F-12 nutrient mixture (1:1) (Mediatech Inc., Herndon, VA) with 10% (wt/vol) charcoal-stripped serum and antibiotics. After an initial incubation for 1 h, the medium was removed along with free-floating cells, and cells that adhered to the culture dishes were cultured in fresh medium (DMEM-F12, 1:1) containing antibiotics and 5% FBS. Medium was changed every other day until cells achieved 80% confluency. We reported previously that cells isolated by the above-described method were almost 99% pure stromal cells (25).

Treatment of Areg protein in stromal cell culture
Stromal cells were cultured on a secure-slip glass coverslip for the purpose of immunofluorescence staining. To observe the proliferative and apoptotic effects of Areg on stromal cells, this growth factor was added to the 80% confluent stromal cells for 24 h in DMEM-F12 (1:1) medium containing 1% (wt/vol) charcoal-stripped FBS. Areg was initially diluted in PBS and added to the culture medium at a concentration of 10 nM. Control cells were treated with PBS. This dose of Areg was selected based on its ability to stimulate tyrosine phosphorylation of epidermal growth factor receptor (EGFR) in MDCK cells (26). To address whether this dose of Areg was effective in stimulating EGFR phosphorylation in stromal cells, this growth factor was added to the 80% confluent stromal cell dish for 1, 5, and 10 min in DMEM-F12 (1:1). Cells were also treated with equal volume of PBS for 10 min as a control. After Areg and PBS additions, cells were harvested and lysates were prepared for Western blotting.

Cell proliferation assay by 5-bromo-2'-deoxyuridine (BrdU) incorporation
Proliferating cells on coverslips were identified by BrdU incorporation inside the nucleus using a kit from Calbiochem (Darmstadt, Germany) (catalog no. HCS30). Cultured stromal cells with and without Areg treatments were labeled with 10 µM BrdU for 1 h before the termination of culture. Cells were next washed with PBS and fixed in cold acid-ethanol mixture (90 ml ethanol, 5 ml acetic acid, 5 ml water). Endogenous peroxidase was inactivated by 0.3% freshly prepared H2O2 before incubation with denaturing solution for 90 min. After washing, cells were incubated with anti-BrdU mouse monoclonal antibody conjugated with biotin overnight at 4 C. Cells were then washed in PBS and incubated for 1 h at room temperature with streptavidin conjugated with Cy3. During final washing of cells, 5 ml diamino-2-phenylindole (DAPI) were added to the 50 ml PBS for the purpose of staining cell nuclei. Coverslips containing cells were then mounted in a microscope slide using Fluoromount G. Fluorescence images were visualized and captured directly by fluorescence microscopy using Eclipse TS100 (Nikon, Tokyo, Japan) with an X-cite 120 fluorescence illumination system (24).

Apoptosis detection
Coverslips containing stromal cells were processed for terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay using a DeadEnd fluorometric TUNEL system (Promega, Madison, WI) that measured the fragmented DNA of apoptotic cells by catalytically incorporating fluorescein-12-deoxyuridine 5-triphosphate at 3'-OH DNA ends using the terminal deoxynucleoidyl transferase. TUNEL assay was performed according to the manufacturer’s instruction with minor modification. Briefly, cells grown over coverslips were fixed in 10% (vol/vol) formalin for 30 min, washed in water, dehydrated in ascending concentrations [30% (vol/vol), 50% (vol/vol), 70% (vol/vol), 85% (vol/vol), 95% (vol/vol), and 100% (vol/vol)] of ethyl alcohol, washed in xylene and acetone, rehydrated in descending concentrations of ethyl alcohol, and finally washed in PBS before incubation with proteinase K (20 µg/ml) for 5 min. Cells were then washed in PBS, refixed in 10% (vol/vol) formalin for 5 min, and divided into three groups. At the beginning of the experiment, the positive control cells were incubated with deoxyribonuclease (RQ1 RNase-free deoxyribonuclease; Promega; catalog no. M6101) for 5 min and rinsed in water. Next, all three groups (positive, treatment, and negative) were incubated with equilibration buffer for 20 min. Subsequently 250 µl of TUNEL reaction mixture containing fluorescein-12-deoxyuridine 5-triphosphate mixed and TdT was added to cells of the positive and treatment groups for 60 min at 37 C in the dark. For negative control, TdT was omitted from the reaction mixture. The reaction was terminated by transferring all the coverslips to the termination buffer for 15 min. The sections were rinsed in PBS and stained with DAPI. Coverslips were mounted in a microscope slide with Fluoromount G and photographed (24).

BrdU-labeled cell counting
The coverslip stained with BrdU in each group was used for counting cells. At least 10 fields from each coverslip were chosen to count cells under the microscope (x400 magnification). The total number of DAPI-positive and BrdU-positive nuclei were counted in each field. The percentage of BrdU-labeled cells in each field was estimated by multiplying 100 with total number of BrdU-labeled nuclei/total number of DAPI-positive cells.

Western blot analysis
Stromal cells treated with or without Areg were harvested and sonicated by using radioimmunoprecipitation assay buffer containing appropriate protease inhibitor cocktail (Complete mini, Roche, Stockholm, Sweden). After centrifugation at 10,000 rpm, the supernatants were collected and loaded (20 µg protein/lane) onto a 7.5% polyacrylamide minigel. Proteins were electrically transferred for 1 h at 100 V to polyvinylidene membranes (Bio-Rad Laboratories, Inc., Hercules, CA). These membranes were subsequently rinsed with Tris-buffered saline containing 0.1% (vol/vol) Tween 20, blocked for 1 h in Tris-buffered saline containing 0.1% (vol/vol) Tween 20 containing 5% (wt/vol) nonfat milk and probed with either rabbit antiphospho-EGFR (Tyr1173) (1:2000 dilution; Cell Signaling Technology, Inc., Danvers, MA) or rabbit anti-EGFR (1:5000 dilution; Upstate, Lake Placid, NY) at 4 C overnight. Blots were then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Detection of protein was performed using Western blotting luminol reagent kit (Santa Cruz Biotechnology, Santa Cruz, CA) followed by fluorography (16, 22, 26). Blots were stripped and reprobed with goat polyclonal actin (C-11) antibody (Santa Cruz).

Statistical analysis
Experiments were replicated three times with tissues collected from different animals. All data were presented as the mean ± SEM. To compare the difference in band intensities, results obtained in Northern blot hybridization experiments were subjected to statistical analysis (SAS 9.1 program; SAS Institute Inc., Cary, NC) using one-way ANOVA. If overall ANOVA revealed significant differences, then comparisons among groups were performed using Tukey’s studentized range test. For other experiments, the difference between two groups was analyzed by Student’s t test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Uterine expression of Ihh mRNAs, but not Hdc and Areg mRNAs, is regulated by P4
Studies in mice demonstrated that uterine expression of Hdc, Areg, and Ihh genes was regulated by P4, and their expressions were correlated with rising P4 levels in the uterus during early pregnancy (16, 17, 18, 19). We speculated that if P4 regulation of these genes in epithelial cells is important for the uterus, the expression of these will also be regulated by P4 in the hamster uterus. To obtain this information, we began our studies by performing in situ hybridization on uterine sections obtained from ovariectomized hamsters treated with oil, P4, or E2 or P4 plus E2. Northern blot hybridization was also performed to confirm the expression of correct transcripts and determine whether there were any effects of P4 on the expression of these genes in the ovariectomized uterus. These results are presented in Figs. 1-6GoGoGoGoGoGo.


Figure 1
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  		FIG. 1. Cross-sections of ovariectomized (Ovex) hamster uteri treated with vehicle (sesame oil) and P4 (2 mg per hamster) were processed for in situ hybridization to demonstrate cell-specific Ihh mRNA expression. Vehicle-treated animals were killed at 24 h. P4-treated animals were killed at 3, 6, 1, and 24 h. Dark-field images are representative of three similar experiments. le, Luminal epithelium; ge, glandular epithelium; s, stroma.

 

Figure 2
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  		FIG. 2. Northern blot analysis of uterine Ihh, Hdc, and Areg mRNAs in the uterus of ovariectomized hamsters treated with vehicle (sesame oil) and P4 (2 mg per hamster). A, Representative Northern blot analysis. B, Bar diagram showing relative mRNA levels in terms of band intensities. Vehicle-treated animals were killed at 24 h. P4-treated animals were killed at 3, 6, 12, and 24 h. Total uterine RNA (6 µg/lane) samples from each treatment group were separated by formaldehyde-agarose gel electrophoresis, transferred, and UV cross-linked to nylon membranes and hybridized sequentially to 32P-labeled Ihh, Hdc, and Areg probes. Total RNA sample from d 4 mouse uterus was hybridized separately as a control for each gene. The blot was also hybridized with Rpl7, a housekeeping gene, to confirm integrity and almost equal loading and blotting of RNA samples. Acridine orange-stained gel showing 28S and 18S rRNAs are also presented. Bar diagram (B) shows the relative levels of Ihh, Hdc, or Areg mRNAs/Rpl7 mRNA after OD scanning of bands in autoradiograms. Results are representative of three experiments. Different letters over bars indicate significant differences (P < 0.05).

 

Figure 3
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  		FIG. 3. Cross-sections of ovariectomized (Ovex) hamster uteri treated with vehicle (sesame oil) or a single injection of P4 (2 mg per hamster) plus E2 (100 ng per hamster) were processed for in situ hybridization to demonstrate cell-specific Ihh mRNA expression. Vehicle-treated animals were killed at 24 h. P4 plus E2-treated animals were killed at 6, 12, and 24 h. Dark-field images (x200) are representative of three experiments. le, Luminal epithelium; ge, glandular epithelium; s, stroma.

 

Figure 4
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  		FIG. 4. Cross-sections of ovariectomized (Ovex) hamster uteri treated with vehicle (sesame oil) or a single injection of P4 (2 mg per hamster) for 2 d and a combination of P4 and E2 (100 ng per hamster) on the third day were processed for in situ hybridization to demonstrate cell-specific Ihh mRNA expression. Vehicle-treated animals were killed at 24 h. P4 plus E2-treated animals were killed at 6, 12, and 24 h. Dark-field images (x200) are representative of three experiments. le, Luminal epithelium; ge, glandular epithelium; s, stroma.

 

Figure 5
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  		FIG. 5. Sections of ovariectomized (Ovex) hamster and mouse uteri treated with vehicle (sesame oil) or P4 (2 mg per animal), E2 (100 ng per animal), or a combination of P4 plus E2 were processed for in situ hybridization to demonstrate cell-specific Hdc mRNA expression. Vehicle-treated animals were killed at 24 h. P4 and E2-treated animals were killed at 6, 12, and 24 h. The uterine sections from d 4 mice and hamsters were used as controls and hybridized with hamster RNA probes on the same day but in separate slides. Dark-field images (x200) are representative of three experiments. Only one representative section from each treatment group is presented. No hybridization signals were noted in the hormone-treated ovariectomized hamsters. CS, Cross-section; le, luminal epithelium; LS, longitudinal section; ge, glandular epithelium; s, stroma.

 

Figure 6
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  		FIG. 6. Sections of ovariectomized (Ovex) hamster and mouse uteri treated with vehicle (sesame oil) or P4 (2 mg per animal), E2 (100 ng per animal), or a combination of P4 plus E2 injection were processed for in situ hybridization to demonstrate cell-specific Areg mRNA expression. Vehicle-treated animals were killed at 24 h. P4- and E2-treated animals were killed at 6, 12, and 24 h. Uterine sections from d 4 mouse and hamster were used as controls and hybridized with hamster RNA probes on the same day but in separate slides. Dark-field images are representative of three experiments. No hybridization signals were noted in the hormone-treated ovariectomized hamsters. CS, Cross-section; le, luminal epithelium; LS, longitudinal section; ge, glandular epithelium; s, stroma.

 
P4, but not E2, regulates Ihh mRNA expression in the ovariectomized hamster uterus
Control uterine sections from ovariectomized hamsters treated with oil showed near background levels of expression of Ihh mRNA by in situ hybridization. However, a single injection of only P4 to ovariectomized animals up-regulated the expression of Ihh mRNAs, mainly in the luminal epithelium and some in the glandular epithelium (Fig. 1Go). We noted the first up-regulation in Ihh mRNA expression over the oil-treated control at 6 h after P4 injection. Thereafter, Ihh gene expression slowly augmented until 24 h after P4 injection (Fig. 1Go). This pattern of Ihh mRNA expression in response to P4 in ovariectomized animals was also noticed in the Northern blot hybridization experiment (Fig. 2Go, A and B). As previously reported in mice (18, 19), we noted an approximately 2.4-kb transcript of Ihh mRNA in the hamster uterus. Whereas Ihh mRNA expression was at the basal level in uterine RNA obtained from ovariectomized animals treated with oil (control), a single injection of P4 gradually up-regulated the level of its expression from 6 to 24 h (Fig. 2AGo). Mouse d 4 uterine RNA served as a positive control. Integrity of total RNA samples used for Northern blot experiments was ascertained by rehybridizing the blot with Rpl 7. Northern blot results of Ihh when quantified and normalized with matching Rpl7, a house keeping gene (22, 23), also revealed gradual increase in its level after P4 treatment (Fig. 2BGo).

When a single injection of P4 and E2 was given together, the Ihh mRNA expression pattern in uterine luminal epithelial cells as detected by in situ hybridization remained high until 12 h but diminished at 24 h (Fig. 3Go). Similarly, an injection of E2 after three daily injections of P4 reduced the level of Ihh mRNA expression at 24 h, compared with its levels observed at 6 and 12 h (Fig. 4Go), suggesting E2 antagonism of P4-induced Ihh mRNA expression in the ovariectomized hamster. Uterine expression of Ihh in the ovariectomized hamsters was not affected by single injection of E2 alone (data not shown) at any time points from 3 to 24 h of post-E2 treatment.

P4 and E2 do not regulate Hdc and Areg mRNA expression in the ovariectomized hamster uterus
Uterine expression of Hdc (Figs. 2Go and 5Go) and Areg (Figs. 2Go and 6Go) mRNAs in the ovariectomized hamsters was not affected by a single injection of P4 as observed by both the in situ and Northern blot hybridization studies. In situ hybridization demonstrated that the basal mRNA levels of Hdc (Fig. 5Go) and Areg (Fig. 6Go) remained constant in oil-treated controls as well as at various time points from 3 to 24 h after P4 treatment. This dose of P4 was physiologically active in the uterus because we observed increased expression of epithelial Ihh mRNA in the same P4-treated uterine tissues (Fig. 1Go). We also noted increased expression of Hdc and Areg mRNAs in uterine sections by in situ hybridization when ovariectomized mice were treated with this dose of P4 (Figs. 5Go and 6Go). P4 primarily induced Hdc and Areg mRNAs in the epithelial cells of the mouse uterus. Uterine sections from d 4 and 5 of pregnant hamster and mouse were used to ascertain the appropriateness of the in situ hybridization procedure and the use of radioactive Hdc and Areg probes, respectively (Figs. 5Go and 6Go). The absence of uterine Hdc and Areg induction in response to P4 in ovariectomized hamsters was also confirmed by Northern blot hybridization (Fig. 2Go, A and B). As previously reported (16, 17), Northern blot hybridization using 32P-labeled hamster-specific Hdc or Areg antisense cRNA probes also detected an approximately 2.6-kb transcript for Hdc and 1.4 kb for Areg, respectively, in mice. Northern blot results of Hdc and Areg when quantified and normalized to Rpl7 also showed no significant changes in their levels after P4 treatment (Fig. 2BGo).

We next studied the role of E2 alone or a combination of P4 and E2 in induction of Hdc and Areg mRNA expression in ovariectomized uterus by in situ hybridization. As expected, no change in Hdc or Areg mRNA expression patterns was noted in response to either E2 or P4 plus E2 over the time course of treatments relative to those observed in ovariectomized oil-treated controls (Figs. 5Go and 6Go). BecauseP4, E2, and P4 plus E2 treatments to ovariectomized hamsters did not induce either Hdc or Areg in the hamster uterus, we presented only one representative section from only P4 or E2 or P4 plus E2 treatment groups in Figs. 5Go and 6Go. These results suggest that both P4 and E2 are not involved in regulation of Hdc and Areg genes in the uterus of ovariectomized hamsters.

Ihh, Hdc, and Areg are differentially expressed in uterine cells during periimplantation period (d 1–8)
The uterine expression patterns of Ihh, Hdc, and Areg in the mouse suggested that their expressions were correlated with uterine preparation for implantation (16, 17, 18, 19). However, similar studies have not been performed in hamsters. Thus, to evaluate the expression patterns of Ihh, Hdc, and Areg in uterine cells during normal pregnancy, we performed in situ and Northern blot hybridization studies.

Ihh mRNA is expressed in uterine epithelial cells from d 2–5 of pregnancy as detected by in situ hybridization
Because uterine expression of Ihh gene was also regulated by P4 in hamsters, we hypothesized that uterine expression of this gene during early pregnancy in hamsters would follow the similar pattern as reported in mice (18, 19). Northern blot analysis of total RNA showed slow but steady increase of Ihh mRNA expression from d 1 to 4 of pregnancy in hamsters. Thereafter, we observed a decline in Ihh transcript expression from d 5 to 8 of pregnancy (Fig. 7Go, A and B). In situ hybridization showed low levels of Ihh expression in uterine epithelial cells on d 1 of pregnancy (Fig. 8Go). However, Ihh mRNA expression started to get stronger in uterine epithelial cells on d 2 and showed further increase on d 3 and 4 of pregnancy. Ihh expression remained constant in luminal epithelial cells surrounding the implantation chamber and further away from the implantation chamber on d 5 of pregnancy. Thereafter Ihh mRNA expression remained at low levels in any types of uterine and embryonic cells at implantation sites from d 6 to 8 of pregnancy (Fig. 8Go). Day 5 uterine sections hybridized with the sense probe did not show any specific positive signals (data not shown).


Figure 7
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  		FIG. 7. Northern blot analysis of uterine Hdc, Areg, and Ihh mRNAs in the uterus of hamsters from d 1 to 8 of pregnancy. A, Representative Northern blot analysis. B, Bar diagram showing relative mRNA levels in terms of band intensities. The blot was hybridized with Rpl7 to confirm integrity and almost equal loading and blotting of RNA samples. Acridine orange-stained gel showing 28S and 18S rRNAs are also presented. Bar diagram (B) shows the relative levels of Ihh, Hdc, or Areg mRNAs/Rpl7 mRNA after OD scanning of bands in autoradiograms. The results represent one of three similar experiments. Different letters over bars indicate significant differences (P < 0.05).

 

Figure 8
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  		FIG. 8. Sections of hamster uteri from d 1 to 8 of pregnancy were processed for in situ hybridization to demonstrate cell-specific Ihh mRNA expression. Dark-field images are representative of three experiments. Inserts show higher magnification of Ihh mRNA accumulation. le, Luminal epithelium; ge, glandular epithelium; s, stroma; cm, circular muscle; lm, longitudinal muscle.

 
Hdc is expressed in glandular epithelial cells only on d 4 of pregnancy
Because P4 alone is essential for uterine receptivity in hamsters but P4 does not regulate Hdc gene expression in this species, we wondered whether Hdc gene expression would occur in the hamster uterus before implantation. Northern blot analysis of total uterine RNA indicated that the level of Hdc mRNA remained steady at basal level from d 1 to 8 of pregnancy except on d 4 when we observed maximum Hdc mRNA expression (Fig. 7Go, A and B). Next, we speculated that if Hdc was important for uterine receptivity on d 4, it would be through cell type-specific expression. Our results of in situ hybridization showed no specific autoradiographic signals in any uterine cell types during d 1–3 of pregnancy. However, Hdc mRNAs appeared only in the glandular epithelial cells on d 4 of pregnancy (Fig. 9Go). Thereafter no distinct expression of Hdc mRNA was detected over background in any uterine or embryonic cell types from d 5 to 8 of pregnancy (data not shown). These results are consistent with those of Northern blot hybridization.


Figure 9
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  		FIG. 9. Cross-sections of hamster uteri from d 1 to 4 of pregnancy were processed for in situ hybridization to demonstrate cell-specific Hdc mRNA expression. Dark-field images are representative of three experiments. le, Luminal epithelium; ge, glandular epithelium; s, stroma; cm, circular muscle; lm, longitudinal muscle.

 
Hdc, but not Ihh, expression in glandular epithelial cells on d 4 of pregnancy is regulated by the presence of blastocysts inside the uterus
Because Hdc and Ihh mRNAs are both expressed in the d 4 hamster uterus, we explored the possibility of the regulation of these genes by the blastocyst. In situ hybridization studies using uterine sections obtained from d 4 pregnant uteri with or without blastocysts, demonstrated that: 1) Hdc mRNA is expressed in glands of the uterine horn containing blastocysts but not in absence of blastocysts (Fig. 10Go) and 2) Ihh mRNA is expressed in the d 4 uterine horns with or without blastocysts, although the level of expression is somewhat lower in the absence of blastocysts. These results suggest that the presence of a blastocyst inside the uterine horn influences Hdc, but not Ihh, mRNA expression in the d 4 uteri of hamsters.


Figure 10
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  		FIG. 10. Cross-sections from d 4 pregnant uteri with or without blastocysts were processed for in situ hybridization to demonstrate blastocyst regulation of Ihh and Hdc mRNA expression. Dark-field images are representative of three experiments. le, Luminal epithelium; ge, glandular epithelium; s, stroma; cm, circular muscle; lm, longitudinal muscle.

 
Areg is expressed in decidual cells surrounding the implanted embryo in hamsters
Because Areg gene is not regulated by P4 in hamsters, it remained to be examined whether this gene was associated with uterine receptivity and implantation in hamsters. Northern blot hybridization showed that Areg mRNA levels did not exhibit any noticeable change from d 1 to 4 of pregnancy. However, its levels were gradually up-regulated from d 5 to 8 (Fig. 7Go, A and B). These surprising results led us to study the cell type-specific expression of Areg gene in the pregnant hamster uterus by in situ hybridization.

The expression of Areg mRNA was not noticed in any uterine cell-types from d 1 to 4 of pregnancy in hamsters (Fig. 11Go). However, accumulation of Areg mRNA was first noticed in several layers of antimesometrial stromal/decidual cells immediate to the implanted blastocyst, compared with its expression further away from the blastocyst on d 5 of pregnancy. This pattern of expression was maintained in implantation sites on d 6–8 of pregnancy (Fig. 11Go). Areg mRNA expression was mainly localized in decidual cells of primary decidual zone that formed in the immediate vicinity surrounding the embryo. Using hamster-specific Areg probes, we also observed epithelial Areg mRNA expression at the d 5 implantation site of mice. These results suggest that whereas epithelial cells are the source of Areg in mice, stromal/decidual cells are the source of Areg in hamsters.


Figure 11
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  		FIG. 11. Cross-sections of hamster uteri from d 1 to 8 of pregnancy were processed for in situ hybridization to demonstrate cell-specific Areg mRNA expression. Dark-field images (x40) are representative of three experiments. le, Luminal epithelium; ge, glandular epithelium; s, stroma; cm, circular muscle; lm, longitudinal muscle.

 
Uterine Areg gene expression in stromal cells adjacent to the implanting embryo is not regulated by the embryo
Because the onset of the Areg gene expression was observed in uterine stromal cells after the initiation of blastocyst implantation, we next examined whether the expression of this gene is induced by the implanted embryo. When we analyzed the expression pattern of Areg gene in d 6 decidual tissues induced by the embryo (deciduum) or suture (deciduomata), expression of this gene was observed in decidual tissues prepared in both ways (Fig. 12Go). Uterine sections from d 5 pregnant hamsters and mice were used as a positive control. These results suggest that Areg expression in decidual cells is not regulated by the implanting embryo. Perhaps Areg gene expression is induced by any stimuli that transform stromal cells to decidual cells in the uterus.


Figure 12
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  		FIG. 12. Cross-sections of hamster d 6 blastocyst-induced deciduum and suture-induced deciduomata were processed for in situ hybridization to demonstrate cell-specific Areg mRNA expression. Day 5 implantation sites of the hamster and mouse were used as controls. Dark-field images (x40) are representative of three experiments. am, Antimesometrial side; bl, blastocyst; em, embryo; m mesometrial side; pdz, primary decidual zone, sdz, secondary decidual zone.

 
Areg stimulates stromal cell proliferation but not apoptosis
Because Areg gene expression was first observed in decidualizing stromal cells surrounding the implanted blastocyst in hamsters, this growth factor may be involved in either stromal cell proliferation or apoptosis. Thus, to assess the probable functions of Areg at the implantation site, stromal cells from d 4 hamster uteri were isolated, cultured in vitro, and treated with either Areg or PBS for 24 h for cell proliferation and apoptosis studies. As shown in Fig. 13AGo by fluorescence microscopy, we observed no increase in TUNEL staining cells in both treatment groups. However, we noted significant increase in the number and percentage of proliferative (BrdU positive) stromal cells (Fig. 13Go, B and C) in Areg-treated cells over the vehicle-treated cells. These results suggest that Areg is cable of inducing proliferation, but not apoptosis, in stromal cells.


Figure 13
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  		FIG. 13. Effect of Areg on stromal cell apoptosis and proliferation in culture. Cells were treated with 10 nM Areg or PBS (as vehicle) for 24 h. TUNEL assay and BrdU incorporation were performed as described in Materials and Methods. DAPI was used to stain nuclei. A, Apoptotic stromal cells in the presence and absence of Areg. Note only one or two TUNEL-positive cells in each treatment group. B, Proliferating stromal cells in the presence and absence of Areg. Note more BrdU-positive cells in Areg-treated group that the PBS-treated group. C, Percentage change in proliferating stromal cells between two groups. The Areg-treated group showed significant (*, P < 0.05) increase in BrdU-labeled cells over the vehicle-treated group.

 
Areg induces ErbB1 phosphorylation in stromal cells
Because Areg preferentially binds to ErbB1, in the form of ErbB1/ErbB1 homodimers (27), we studied Areg-induced ErbB1 receptor autophosphorylation in stromal cells in culture. After Areg treatment for 1–10 min, cells were harvested, and lysates were prepared for Western blotting. We observed an immediate increase in tyrosine phosphorylated ErbB1 in stromal cells within 1 min of Areg exposure over the vehicle-treated control cells. This Areg-induced ErbB1 phosphorylation remained elevated until 10 min (Fig. 14Go). We did not observe any noticeable change in total EGFR levels in both the Areg- and vehicle-treated cells (Fig. 14Go). Thus, a rapid ErbB1 phosphorylation is probably a requirement for Areg-induced stromal cell proliferation.


Figure 14
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  		FIG. 14. Western blot analysis of EGFR and phospho EGFR in Areg-treated uterine stroma cell in primary culture. Uterine stroma cells were treated with 10 nM of Areg. Cells were harvested after 1, 5, and 10 min of Areg treatment (vehicle was treated with PBS for 10 min). Two separate blots were made using the same samples and equal amount of proteins and immunoblotted with the primary antibody for EGFR, phosphor-EGFR, and actin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The signal that transforms the uterus to the receptive stage for initiating events involved in blastocyst implantation is not understood. So far, what is established is that whereas both the ovarian steroid hormones P4 and E are required for uterine receptivity and implantation in mice and rats (2, 3), P4 alone is needed in hamsters, pigs, rabbits, guinea pigs, and monkeys (5, 6, 7, 8, 9, 10, 11, 12, 13, 14). Because the majority of species including perhaps humans in whom luteal P4, but not E, is a requirement for implantation (15), little effort has been given to identify the downstream genes involved in P4 functions in these species. In an attempt to obtain information on P4 actions in the uterus of species in which ovarian P4 is the sole requirement for uterine receptivity, we evaluated whether the three well-established P4-regulated and implantation-specific uterine genes, Ihh, Hdc, and Areg, in mice (16, 17, 18, 19) are also regulated by P4 and involved in uterine receptivity and implantation in the hamster. Our study established, for the first time, distinct species differences in the expression patterns and hormonal regulations of Hdc and Areg, but not Ihh, in the hamster when compared with mice (16, 17, 18, 19). P4 stimulation of Ihh and the gradual increase in Ihh mRNA expression from d 2 to 5 of pregnancy in the hamster uterus is similar to the pattern of expression in mice as reported by two separate investigators (18, 19). However, uterine expression of mRNAs for both Hdc and Areg genes were not regulated by ovarian hormones in hamsters. During d 1–8 of pregnancy in hamsters, whereas Hdc mRNA expression was observed only on d 4 of pregnancy and only in uterine glandular epithelial cells, Areg mRNA expression was noted in decidual cells surrounding the implanted embryos.

The blastocyst initiates the implantation process in an appropriately prepared receptive uterus. Uterine development to this receptive state is primarily influenced by P4. P4 actions in the uterus are normally mediated via binding to nuclear P4 receptor (PR) (reviewed in Ref. 18). The human and mouse PR consists of two main isoforms, PR-A and PR-B. Evidence also exists for other PR isoforms such as PR-C, PR-S, and PR-T (reviewed in Ref. 28). Whereas PR-A functions as a transcriptional inhibitor of PR-B, PR-C may act as a modulator of PR-A and PR-B transcriptional activities in those cells that produce these isoforms (28). In mice, the importance of uterine PR in the establishment of pregnancy has been studied by evaluating uterine PR mRNA expression patterns during early pregnancy as well as deletion of the PR gene (29). Ablation of PR (A/B) or just PR-A resulted in infertility in females due to ovulation, implantation, and decidualization problems, whereas deletion of PR-B did not affect biological responses of the ovary and uterus to P4 (reviewed in Ref. 30). However, the uterine PR mRNA expression patterns have not been studied in hamsters. Previous studies using P4-binding assays suggested that there is a specific PR in the cyclic hamsters that increases as serum estrogen levels increased (31). However, how P4/PR-mediated functions are executed in the uterus is not clearly understood. Based on our current knowledge in mice, preparation of the uterus for implantation is regulated by many signaling molecules of the growth factor, cytokine, tissue morphogen, and inflammatory gene families (reviewed in Refs. 32 and 33). Several members of these families were also reported to be regulated by P4 in mice. Considering the essential influence of P4 in uterine preparation for implantation in all species so far studied, we suspected that hormonal regulation and expression of P4-regulated gene families in the hamster uterus may mirror the regulation and expression patterns of these genes as observed in the mouse uterus.

Ihh is a member of the developmentally regulated morphogens, the hedgehog gene family. Two other members of this family include Sonic hedgehog and Desert hedgehog (reviewed in Ref. 18). These molecules actively participate in epitheliomesenchymal cross talk, regulating both epithelial cell proliferation and differentiation (reviewed in Ref. 34). The work presented here shows that Ihh is produced only by the epithelial cells of the uterus. Its expression initiated on d 2 of pregnancy when the cyclic uterus first shows pregnancy-associated remodeling in response to mating and fertilization on the estrus day (24). The gradual increase in its expression from d 2 to 5 is suggestive of its direct involvement in day-to-day preparatory changes of the uterus for implantation. It is possible that Ihh induces differentiation of uterine epithelial cells by ending their proliferation. It is also intriguing to consider that Ihh may be involved in inducing proliferation of stromal cells because Ihh protein is diffusible (19). In this regard, it has been demonstrated in mice that whereas Ihh is up-regulated in the luminal epithelium during uterine preparation for implantation on d 3 and 4, its downstream signaling molecules Ptc, Gli1, and Gli2 are also strongly up-regulated in the uterine stroma (18, 19). Because uterine receptivity in hamsters can be achieved by P4 only, regulation of Ihh gene by P4, but not by the blastocyst, is anticipated. Indeed, by injecting P4 and E2 separately to ovariectomized hamsters, we observed regulation of Ihh gene in the uterus by P4, but not by E2. However, whether P4-induced Ihh expression is mediated via PR receptors is an open question in hamsters. This has not been tested because PR antagonists such as RU 486 do not block PR-mediated P4 actions in hamsters (35). It has been demonstrated by competitive binding assay that RU 486 did not compete with hamster uterine PR because the hamster PR hormone binding domain has a cysteine in the place of a glycine (35, 36). After initiation of implantation, expression of Ihh was completely abolished from the uterine tissues, suggesting that Ihh action is necessary in the uterus only during the preparatory phase for implantation but not during the decidualization phase.

When we investigated Hdc gene regulation by steroid hormones, we noted that the expression of this gene in ovariectomized hamsters was not regulated by P4 or E2 or combined treatment of P4 and E2. These unanticipated data suggest that Hdc may not be important for various hormone-regulated events during uterine preparation for implantation. To our surprise, however, when we examined the location of Hdc mRNAs in the uterus during early pregnancy, we found that Hdc was expressed only in d 4 uterus and only in the glandular epithelium. The absence of Hdc expression on the other days of pregnancy and in the luminal epithelium is surprising because we previously reported its expression both in the glandular and luminal epithelial cells from d 1–8 pregnancy in mice (16). By analyzing the expression patterns of Hdc gene between mice and hamsters, we noted one similarity in its expression on d 4 when the uterus is maximally receptive for implantation. These data suggest that Hdc is in some way important in the uterus on d 4. Because Hdc-null mice showed no problems in uterine preparation and implantation (37), Hdc may play cooperative roles with other factors on this day. Our observation that steroid hormone did not alter uterine Hdc in the hamster uterus suggests that its expression in the d 4 receptive uterus of this species is possibly intertwined with some other regulators of uterine functions. In this regard, it is also unknown whether the presence of blastocysts inside the d 4 uterus is somehow involved in the induction of Hdc gene in uterine glands. Indeed, we observed that Hdc is induced in glandular epithelial cells of the blastocyst-containing uterine horn but not in the blastocyst-free uterine horn. These results suggest that Hdc expression in the d 4 uterus is influenced by certain blastocyst-derived factors. These blastocyst-derived factors are presumably not P4 and E because they do not stimulate uterine expression of Hdc. Thus, this embryonic signal(s) remains to be determined.

We previously reported that uterine Areg is regulated by P4, and its expression could serve as a molecular maker for the receptive state of the uterus due to its transient expression in epithelial cells on d 4 of pregnancy in mice (17). With the initiation of implantation, its expression in the luminal epithelium surrounding the implanting blastocyst remained strong, whereas it is reduced in the luminal epithelium further away from the blastocyst. Areg expression is not observed in the implantation sites after d 6 of pregnancy in mice. The work presented here showed no hormonal regulation of uterine Areg in hamsters. Moreover, we did not observe any relationship between Areg expression and uterine receptivity in the hamster because it was not expressed in the uterus during its receptivity for implantation. Interestingly, however, its expression was observed only in decidual cells adjacent to the implanting embryos from d 5 to 8 of pregnancy, suggesting its involvement in stromal cell proliferation and decidualization processes in hamsters. Previously we reported that whereas HB-EGF is important for stromal cell decidualization at the implantation site of mice, it is not important in hamsters (22). Here we report a reverse scenario: whereas Areg is not involved in the uterine decidualization process in mice, it is important in hamsters. Because both HB-EGF and Areg are potent stimulators of EGFR phosphorylation and both can bind heparin (17, 21, 22, 26), it is possible that their functions are complementary depending on the species. Because the induction of Areg at the implantation site and its lack of regulation by steroids are unexpected findings, we wondered whether expression of this gene at the implantation site was regulated by the blastocyst. Expression of Areg in suture-stimulated deciduomal tissues suggests that Areg expression in decidual tissues is also not induced by the blastocyst. Thus, it is possible that any stimulus that triggers stromal decidualization and local inflammatory reactions may induce Areg expression in the uterus.

The initiation of the implantation process stimulates the proliferation of stromal cells that lie just underneath the implanting blastocyst. At the beginning of implantation, Areg gene expression was mainly observed in proliferating stromal cells. Thus, it is possible that one of the possible functions of Areg may be to influence the proliferation of stromal cells. Indeed, in an in vitro primary uterine stromal cell culture system, we observed ErbB1-mediated Areg-induced stromal cell proliferation but not apoptosis. It has been reported previously that Areg mediates its actions via the EGFR ErbB1 (26, 27). In this regard it is noteworthy that ErbB1 gene is expressed in stromal cells surrounding the implanted blastocyst in hamsters (Wang, X., and B. C. Paria, unpublished results).

In conclusion, when we compared our results of Ihh, Hdc and Areg gene expressions during early pregnancy and their hormonal regulations in hamsters with the previously reported data in the mice, we noted the presence of similarities and dissimilarities among species. Whereas uterine Ihh expression and its hormonal regulation showed similar patterns in both the species, distinct differences were noted in the uterine expression and hormonal regulations of Hdc and Areg between these two species. Therefore, caution should be used when drawing conclusions about the importance of a molecule for specific events based on studies performed in a single species. Our data highlight how comparison studies among species may help to identify common signaling systems that may potentially be important in understanding uterine receptivity and implantation in humans.


   Acknowledgments
 
We acknowledge the support from all members of the Division of Reproductive and Developmental Biology of the Department of Pediatrics. The support of the National Cooperative Program on Trophoblast-Maternal Tissue Interactions is gratefully acknowledged. Our special thanks to S. K. Dey and S. K. Das for allowing free access to their laboratories and Hehai Wang for statistical analysis.


   Footnotes
 
This work was supported by National Institutes of Health Grants HD044741 and UO1 HD042636 (to B.C.P.).

Disclosure summary: all authors have nothing to declare.

First Published Online June 22, 2006

Abbreviations: Areg, Amphiregulin; BrdU, 5-bromo-2'-deoxyuridine; DAPI, diamino-2-phenylindole; E, estrogen; E2, estrodiol-17ß; EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; HB-EGF, heparin-binding epidermal growth factor-like growth factor; HBSS, Hanks’ balanced salt solution; Hdc, histidine decarboxylase; Ihh, Indian hedgehog; P4, progesterone; PR, P4 receptor; RNase, ribonuclease; TdT, terminal deoxynucleotidyl transferase; TUNEL, TdT-mediated deoxyuridine triphosphate nick end labeling.

Received February 22, 2006.

Accepted for publication June 9, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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