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

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. 1-6.

View larger version (113K): [in this window] [in a new window]   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.

View larger version (45K): [in this window] [in a new window]   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).

View larger version (120K): [in this window] [in a new window]   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.

View larger version (126K): [in this window] [in a new window]   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.

View larger version (120K): [in this window] [in a new window]   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.

View larger version (120K): [in this window] [in a new window]   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.

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. 3). 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. 4), 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. 2 and 5) and Areg (Figs. 2 and 6) 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. 5) and Areg (Fig. 6) 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. 1). 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. 5 and 6). 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. 5 and 6). The absence of uterine Hdc and Areg induction in response to P₄ in ovariectomized hamsters was also confirmed by Northern blot hybridization (Fig. 2, 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. 2B).

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. 5 and 6). 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. 5 and 6. 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. 7, A and B). In situ hybridization showed low levels of Ihh expression in uterine epithelial cells on d 1 of pregnancy (Fig. 8). 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. 8). Day 5 uterine sections hybridized with the sense probe did not show any specific positive signals (data not shown).

View larger version (56K): [in this window] [in a new window]   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).

View larger version (130K): [in this window] [in a new window]   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.

View larger version (113K): [in this window] [in a new window]   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.

View larger version (87K): [in this window] [in a new window]   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.

The expression of Areg mRNA was not noticed in any uterine cell-types from d 1 to 4 of pregnancy in hamsters (Fig. 11). 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. 11). 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.

View larger version (100K): [in this window] [in a new window]   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.

View larger version (103K): [in this window] [in a new window]   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.

View larger version (23K): [in this window] [in a new window]   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.

View larger version (63K): [in this window] [in a new window]   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 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

 

1. Krehbiel RH 1941 The effects of theelin on delayed implantation in the pregnant lactating rat. Ant Rec 81:381[CrossRef]
2. Psychoyos A 1973 Endocrine control of egg implantation. In: Greep RO, Astwood EG, Geiger SR, eds. Handbook of physiology. Washington, DC: American Physiological Society; 187–215
3. McCormack JT, Greenwald GS 1974 Evidence for a preimplantation rise in estradiol-17ß levels on day 4 of pregnancy in the mouse. J Reprod Fertil 41:297–301[Abstract/Free Full Text]
4. Yoshinaga K, Adams CE 1966 Delayed implantation in the spayed progesterone treated adult mouse. J Reprod Fertil 12:593–595[Abstract/Free Full Text]
5. Prasad MRN, Orsini MW, Meyer RK 1960 Nidation in progesterone-treated, estrogen-deficient hamsters, Mesocricetus auratus (Waterhouse). Proc Soc Exp Biol Med 104:48–51[Medline]
6. Harper MJK, Dowd D, Elliot ASW 1969 Implantation and embryonic development in the ovariectomized-adrenalectomized hamster. Biol Reprod 1:253–257[Abstract]
7. Orsini MW, Meyer RK 1962 Effects of varying doses of progesterone on implantation in the ovariectomized hamster. Proc Soc Exp Biol Med 110:713–715
8. George FW, Wilson JD 1978 Estrogen formation in the early rabbit embryo. Science 199:200–201[Abstract/Free Full Text]
9. Hoversland RC, Dey SK, Johnson DC 1982 Aromatase activity in the rabbit blastocyst. J Reprod Fertil 66:259–263[Abstract/Free Full Text]
10. Heap RB, Flint APF, Gadsby JE 1981 Embryonic signals and maternal recognition. In: Glasser S, Bullock DW, eds. Cellular and molecular aspects of implantation. New York: Plenum Press; 311–325
11. Perry JS, Heap RB, Amoroso EC 1973 Steroid hormone production by pig blastocysts. Nature 245:45–47[CrossRef][Medline]
12. Deanesly R 1960 Normal implantation in ovariectomized guinea pigs. Nature 186:327–328[Medline]
13. Meyer RK, Wolf RC, Arslan M 1969 Implantation and maintenance of pregnancy in progesterone treated ovariectomized monkeys (Macaca mulatta). In: Recent advances in primatology. Vol 2. Basel: S Karger; 30–35
14. Ghosh D, De P, Sengupta J 1994 Luteal phase ovarian estrogen is not essential for implantation and maintenance of pregnancy from surrogate embryo transfer in the rhesus monkey. Hum Reprod 9:629–637[Abstract/Free Full Text]
15. Zegers-Hochschild F, Altieri E 1995 Luteal estrogen is not required for the establishment of pregnancy in the human. J Assist Reprod Genet 12:224–228[CrossRef][Medline]
16. Paria BC, Das N, Das SK, Xhao X, Dileepan KN, Dey SK 1998 Histidine decarboxylase gene in the mouse uterus is regulated by progesterone and correlates with uterine differentiation for blastocyst implantation. Endocrinology 139:3958–3966[Abstract/Free Full Text]
17. Das SK, Chakraborty I, Paria BC, Wang X-N, Plowman G, Dey SK 1995 Amphiregulin is an implantation-specific and progesterone-regulated gene in the mouse uterus. Mol Endocrinol 9:691–705[Abstract]
18. Takamoto N, Zhao B, Tsai SY, DeMayo FJ 2002 Identification of Indian hedgehog as a progesterone-responsive gene in the murine uterus. Mol Endocrinol 16:2338–2348[Abstract/Free Full Text]
19. Matsumoto H, Zhao X, Das SK, Hogan BLM, Dey SK 2002 Indian hedgehog as a progesterone-responsive factor mediating epithelial-mesenchymal interactions in the mouse uterus. Dev Biol 245:280–290[CrossRef][Medline]
20. Ingham PW, McMahon AP 2001 Hedgehog signaling in animal development: paradigms and principles. Genes Dev 15:3059–3087[Free Full Text]