Regulation of tryptophan 2,3-dioxygenase by HOXA10 enhances embryo viability through serotonin signaling

Tryptophan 2,3-dioxygenase (TDO) is expressed in endometrium and catabolizes tryptophan, a precursor in the biosynthesis of serotonin. Tryptophan metabolism is an important mechanism for regulation of serotonin levels. Preimplantation mouse embryos are known to express serotonin receptors, specifically the 5-HT1D and 5-HT7 serotonin receptor subtypes. Here we demonstrate that Hoxa10 regulates endometrial TDO expression and improves embryo viability through increased serotonin production. Transfection of pcDNA-Hoxa10 to the murine uterus increased total TDO expression. In vitro, epithelial cell TDO expression was decreased after transfection with Hoxa10. Decreased glandular TDO in response to HOXA10 may augment serotonin production by increasing tryptophan availability. Conversely, stromal TDO expression increased with constitutive Hoxa10 expression. In mice, epithelial serotonin was increased in response to constitutive expression of Hoxa10. Embryo quality was impaired after treatment with Hoxa10 antisense. Blockade of serotonin receptors 1D and 7 also resulted in impaired embryo development, indicating an essential role for Hoxa10 induction of TDO and subsequent serotonin production in embryo development. Transfection of pcDNA-TDO also decreased the number of T cells in the endometrial stroma. We have shown a novel mechanism by which HOXA10 regulates endometrial TDO expression. In the endometrial stroma, HOXA10 increases TDO mRNA, which may increase tryptophan catabolism, allowing for immune tolerance at the time of embryo implantation. In endometrial glands, HOXA10 decreases TDO mRNA leading to increased serotonin that in turn acts to promote normal embryo development.

tryptophan degradation, implantation, serotonin the induction of receptivity in the mammalian endometrium requires specific molecular and structural alterations in response to hormonal signals. These changes occur within the glandular epithelium and the endometrial stroma to create a favorable environment for embryo implantation. The specific genes responsible for inducing endometrial receptivity, as well as their regulation and function at the time of implantation, remain incompletely characterized. Hoxa10 is a member of the homeobox (Hox) gene family, which encodes transcription factors that function to control embryonic development and to regulate gene expression within the endometrium during the menstrual cycle (39). Hox genes expressed within the reproductive tract regulate normal developmental patterning during the embryonic period and continue to function in the adult. Hoxa10 is expressed by the endometrial glands and stroma throughout the menstrual cycle and its expression is regulated by estradiol and progesterone (36, 38). Mice with targeted disruption of either Hoxa10 or Hoxa11 are infertile due to abnormal endometrial receptivity but are able to produce viable embryos (6, 16, 32). There are no known human mutations of the Hoxa10 or Hoxa11 genes; however, women with implantation defects, including endometriosis, polycystic ovarian syndrome, hydrosalpinges, and submucosal myomas, have diminished expression of these genes (9, 12, 30, 37). Endometrium from women using contraception have also shown to have decreased levels of Hoxa10 (41). The associations between altered Hoxa10 expression and implantation defects are clear. The specific mechanisms by which HOXA10 functions in endometrial receptivity are not fully characterized. HOXA10 functions as a transcription factor and several of its target genes have recently been identified. HOXA10 acts as a transcriptional repressor of Emx2, which is expressed during the embryonic period in the developing female reproductive tract as well as within the adult endometrium (43). Endometrial β3-integrin subunit gene expression is directly upregulated by HOXA10 (13). cDNA microarray has recently been used to broadly search for potential downstream targets of HOXA10 (24, 45). When HOXA10 was overexpressed, several target genes were found to have significantly increased expression. Of these, the largest increase was in the tryptophan 2,3-dioxygenase (TDO) gene that increased eightfold in response to constitutive expression of HOXA10 (45). TDO is one of two mammalian tryptophan degrading enzymes; the second enzyme is indolamine 2,3-dioxygenase (IDO; Ref. 42). Tryptophan is an essential amino acid that is an immediate precursor in the biosynthesis of serotonin. Serotonin synthesis is dependent on tryptophan availability; thus increased tryptophan degradation decreases serotonin (27, 40). Inhibition of TDO activity, leading to an increase in available tryptophan, increases serotonin levels (31). TDO is ubiquitously expressed and has been found in many tissues including liver, brain, skin, and uterus. TDO demonstrates a menstrual cycle dependent expression pattern in the endometrium and is induced in decidualized stromal cells at the time of embryo implantation (35). IDO is expressed by trophoblasts but not by endometrial cells (4). Tryptophan degradation occurs within the murine embryonic and extra-embryonic tissues, peaking on day 6.5 postcoitum. This activity coincides with high expression levels of TDO in the endometrium (26, 34). T-cell proliferation is inhibited by tryptophan degradation. Tryptophan degradation by IDO is necessary for embryo implantation (28). Inhibition of IDO function leads to embryo rejection by T lymphocytes. IDO is expressed by trophoblasts, after TDO expression in the endometrium (21, 34). Mice with targeted disruption of Hoxa10 have an accumulation of T lymphocytes within the endometrial stroma (46). This suggests a link between HOXA10 and regulation of immune tolerance, possibly through TDO. Serotonin [5-hydroxytrytamine (5-HT)] is a hormone and neurotransmitter with diverse effects. Its actions have been best characterized within the central nervous system, vasculature, and gastrointestinal tract. Serotonin has also been identified within the female reproductive tract in ovaries, uterus, oviducts, placenta, and ovarian follicular fluid (1, 2, 7, 8, 10, 18, 25). At least 15 serotonin receptors, grouped into 7 families due to their signaling mechanisms, have been cloned (20). In mammals, multiple serotonin receptor subtypes are located within the reproductive tract (15). Preimplantation mouse embryos express the serotonin receptors 5-HT1D and 5-HT7 (3, 17, 44). In vitro exposure of embryos to serotonin at 1 μM reduced mean cell number, while lower concentrations did not alter embryo cell numbers (17). Maternally derived serotonin, however, is necessary for normal embryo development. Female mice with targeted disruption of tryptophan hydroxylase 1, the rate-limiting enzyme in serotonin synthesis, produce offspring with abnormal central nervous system development, regardless of the genotype of the fetus (11). These studies show that regulation of maternally derived serotonin levels is necessary for normal embryo development. TDO expression is upregulated in uteri of mice overexpressing Hoxa10; we therefore sought to examine the cell-specific regulation of TDO by HOXA10. Because of the importance of available tryptophan in serotonin synthesis, we examined serotonin localization within the endometrium in mice with altered expression of HOXA10. Finally, we examined embryos of mice treated with serotonin receptor antagonists to determine the direct effects serotonin on embryo viability.

Cell culture and transfection of cell lines. The human endometrial stromal cell line HESC, the primary third trimester human decidual cell (HDC), and the human endometrial adenocarcinoma cell line Ishikawa were used. HESC cells are a well-characterized telomerase immortalized human endometrial stromal cell line that is phenotypically and morphologically similar to parent cells (14, 19, 29). Third trimester human decidual cells were extracted from women undergoing repeat cesarean sections at term (23). HESC and HDC cells were maintained in a DMEM solution (Sigma, St. Louis, MO) supplemented with 10% charcoal stripped calf serum, 1% penicillin/streptomycin solution, and 1% sodium pyruvate. Ishikawa cells were maintained in a MEM solution (Sigma) supplemented with 10% charcoal stripped calf serum, 1% penicillin/streptomycin solution, and 1% sodium pyruvate. HOXA10 cDNA cloned into the pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA) and the pcDNA3.1(+) vector alone have been described previously (5). HESC, HDC, and Ishikawa cells, grown to 60–70% confluence, were transfected using Lipofectamine 2000 (Invitrogen) with either pcDNA3.1(+)/HOXA10 cDNA (1 μg/ml) or empty pcDNA3.1(+) vector (1 μg/ml). Human decidual cells were also transfected with pcDNA3.1(+)/HOXA10 antisense. In vivo gene transfection. Nulliparous CD1 male and female mice (8–12 wk old; Charles River, Wilmington, MA) were mated and examined every 12 h until the presence of a vaginal plug. Presence of a vaginal plug designated day 1 postcoitum. Twenty-four to thirty hours after detection of the vaginal plug, 13 female mice were anesthetized with 200–400 μl of a 10% ketamine-5% xylazine solution intraperitoneally in accordance with Yale Animal Care and Use Committee approved animal care protocols. A midline abdominal incision was made with a scalpel to gain exposure to the uterus. DNA/liposome complexes were prepared using previously titrated ratios of DNA to liposome (5). A final concentration of 16 μg/ml of DNA and 40 μg/ml of Lipofectamine [a 3:1 (wt/wt) liposome formulationof the polycationic lipid (DOPSA) and the neutral lipid (DOPE) in membrane filtered water; Invitrogen] was prepared in Opti-MEM reduced serum media (Invitrogen) to a total volume of 75 μl per animal. Twenty-five microliters of of complexed DNA/liposome mixture containing either HOXA10 cDNA/pcDNA3.1(+) (n = 5), HOXA10 antisense cDNA/pcDNA3.1(+) (n = 5), TDO cDNA/pcDNA3.1(+) (n = 4), or empty pcDNA3.1(+) vector (n = 10) were injected into the base of each uterine horn using a 28-gauge U-100 insulin syringe (total volume injected for each animal = 50 μl). Both uterine horns were treated similarly to ensure exposure of the entire uterus to the treatment vector. Peritoneal closure was performed with a single running suture of 4–0 Polysorb suture (Covidien Syneture, Mansfield, MA). Skin was closed with interrupted stitches using the same suture. Postoperative care was carried out according to the Yale Animal Care and Use Committee guidelines. In vivo serotonin antagonist treatment. Nulliparous female CD1 mice (8–12 wk old) were treated with 5 IU of pregnant mare's serum (Sigma). Forty-eight hours later, ovulation was triggered with 5 IU of human chorionic gonadotropin (Sigma). Animals were then mated with male CD1 mice and examined 12 h later for the presence of a vaginal plug. The presence of a vaginal plug designated day 1 postcoitum 48 h after detection of a vaginal plug, six pregnant female mice were anesthetized, and laparotomy was performed as described above. M2 medium (Sigma) containing a 1 mM concentration of both the 5-HT 1D receptor antagonist GR127935 and the 5-HT 7 receptor antagonist SB269970 (Sigma) was used for timed intrauterine injection. Three mice were treated with 25 μl of the M2 solution containing 1 mM of serotonin receptor antagonists injected into the base of each uterine horn. Three control mice were treated with 25 μl of M2 medium alone. Abdominal closure and postoperative care were performed as above. Provocation of inflammatory response within the endometrium. Control mice (n = 4) and treatment mice (n = 4) were randomly assigned to in vivo transfection. All mice were mated with vasectomized males to induce a pseudopregnant state. The presence of a vaginal plug designated day 1 postcoitum 48 h after detection of a vaginal plug, eight pregnant female mice were anesthetized, and laparotomy was performed as described above. Treatment mice were transfected with TDO cDNA/pcDNA3.1(+) plasmid, and control mice were transfected with empty plasmid vector according to our in vivo transfection protocol (previously described above). Twenty-four hours after transfection, 100 μl of sesame oil were injected intravaginally to both control and TDO-plasmid-treated mice to provoke a decidual response within the endometrium. Twenty-four hours later, mice were euthanized by cervical dislocation and uteri were collected for immunohistochemistry. Tissue dissection and embryo collection. Mice were euthanized on day 4.5 postcoitum (48 h after laparotomy) in accordance with the Yale Animal Care and Use Committee protocol. Uteri were carefully dissected using sterile technique. Uteri from six mice [2 control, 2 HOXA10/pcDNA3.1(+), and 2 HOXA10 antisense/pcDNA3.1(+)] were immediately frozen at −80° C. Uteri from an additional 13 mice [4 control pcDNA3.1(+), 3 HOXA10 antisense/pcDNA3.1(+), 3 M2 medium, and 3 M2 medium plus 5-HT receptor antagonist] were used for embryo flushing. Embryos were flushed by infusing 200 μl of M2 medium with a 28-gauge needle inserted into the caudal portion of the uterus, thus flushing the embryos out through each uterine horn. Examination of embryo number and quality (percentage of blastocysts recovered) was then performed using a dissecting microscope. Quantitative RT-PCR. Total RNA was isolated from cultured cells 48 h after transfection using the RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. Total RNA was extracted from murine uteri by using Trizol reagent in accordance with the manufacturer's guidelines (Life Technologies, Gaithersburg, MD). Five hundred nanograms of total RNA were reverse transcribed in 20 μl of reaction mixture using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Quantitative real-time RT-PCR reactions were prepared using the iQ SYBR Green Supermix (Bio-Rad). The conditions for PCR were optimized analyzing the melting curves of the products acquired. Amplification efficiency was assessed after varying primer annealing temperatures, primer concentrations, template concentrations, and cycle number. All products were analyzed in the linear phase of amplification. Each PCR reaction consisted of the following: 1 μl of cDNA template, 1 μl of forward primer (1 uM), 1 μl of reverse primer (1 μM), 9.5 μl of nuclease-free H20, and 12.5 μl of iQ SYBR Green Supermix. TDO intron-spanning primers were designed using PerlPrimer version 1.1.14. HOXA10 and β-actin primers have been previously described (33). TDO human primers were forward 5′-CTTAGTAAAGGTGAAAGACGG-3′ and reverse 5′-GTCCATAAGAGAAGTCAGCA-3′. TDO mouse primers were forward 5′-CATGGCTGGAAAGAACACCT-3′ and reverse 5′-TTCTTCTTCCAGGCCTTTCA-3′. The Bio-Rad iCycler iQ system (Bio-Rad) was used to quantify fluorescence of PCR products during amplification. RT-PCR reactions were performed for 1 cycle at 95°C for 3 min, followed by 40 cycles at 95°C for 15 s and 59 °C for 20 s. Melting curve data were collected for analysis. Relative gene expression was determined by analyzing data using the 2−ΔΔCT method to adjust for expression of β-actin (22). All experiments were conducted in triplicate. Samples without cDNA template were used as negative controls. Serotonin immunohistochemistry. Serotonin (5-HT) localization was evaluated by immunohistochemistry using a goat monoclonal antibody to Serotonin (20079; Immunostar, Hudson, WI). Specimens were embedded in paraffin, cut into serial 5-μm sections, and mounted on slides. The sections were deparaffinized and dehydrated in xylene and ethanol. After a 5-min rinse in distilled water, an antigen-presenting step was performed by steaming the slides in 0.01 M sodium citrate buffer for 20 min and cooling for 20 min. The slides were then rinsed for 5 min in PBS-Tween (PBST), and sections were circumscribed with a hydrophobic pen. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. After incubation with 1.5% normal horse blocking serum in PBST for 1 h, the sections were incubated overnight at 4° C with primary antibody (anti-Serotonin in a 1:20,000 dilution). Normal goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA) was used as a negative control. The sections were then rinsed in PBST and incubated with biotinylated horse anti-goat secondary antisera (Vector Laboratories, Burlingame, CA) for 60 min. Slides were then rinsed in PBST and incubated in avidin and biotinylated peroxidase (ABC elite, Vector Laboratories) for 15 min. After being rinsed in PBST, the slides were then incubated in diaminobenzidine for 5 min. Slides were then rinsed in water. Hemotoxylin was used for counterstaining. Slides were then rehydrated via a series of ethanol and xylene washes, and coverslips were mounted using Permount. CD3 immunohistochemistry. Specimens were imbedded in paraffin, cut into 5-μm sections, and mounted on slides. The sections were deparaffinized and dehydrated in xylene and ethanol. After a 5 min rinse in distilled water, an antigen-presenting step was performed by steaming the slides in 0.01 M sodium citrate buffer for 20 min and cooling for 20 min. The slides were then rinsed for 5 min in PBST, and sections were circumscribed with a hydrophobic pen. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. After incubation with 1.5% normal goat blocking serum in PBST for 1 h, slides were incubated overnight at 4° C with a 1:200 dilution of mouse CD3 antibody (sc-20047; Santa Cruz Biotechnology). Normal mouse IgG (Santa Cruz Biotechnology) was used as negative control. Slides were then rinsed in PBST and incubated in 3.5 μg/ml of biotinylated goat anti-mouse (Vector Laboratories) for 1 h at 4° C. After being rinsed in PBST, the slides were incubated in ABC elite (Vector Laboratories) for 15 min at room temperature. Slides were then washed with PBST and incubated for 5 min in diaminobenzidine (Vector Laboratories). Slides were then rinsed in water. Hemotoxylin was used for counterstaining. Slides were then dehydrated via a series of ethanol and xylene washes, and coverslips were mounted using Permount. Three sections per uterus were examined. Three high-powered fields were examined per section. The number of CD3+ cells per high-power field (×600) were counted and averaged for each animal (n = 8). Statistical analysis. Quantitative PCR and CD3+ staining results were compared using Student's t-test via SPSS software (version 12.0.1). Embryo number was compared using the Mann-Whitney U-test via SPSS software (version 12.0.1). . Embryo quality (reflected as percent blastocysts) results were compared using independent samples t-test via SPSS software (version 12.0.1). P < 0.05 was considered statistically significant.

TDO expression in murine uteri. We (45) have previously shown that TDO expression increases eightfold in murine uterine tissue of mice overexpressing Hoxa10. We confirmed this microarray data by examining TDO expression in whole uteri of mice treated with Hoxa10/pcDNA3.1(+), Hoxa10 antisense/pcDNA3.1(+), and controls [empty pcDNA3.1(+)]. Mice treated with Hoxa10-plasmid in vivo gene transfection showed a 3.8-fold increase in uterine TDO mRNA expression compared with controls (P < 0.05). TDO mRNA expression was not significantly altered in mice treated with Hoxa10 antisense. (Fig. 1A). TDO expression in endometrial cell lines. Because whole uterine tissue represents multiple cell types, we sought to quantify TDO expression in individual cell lines. To confirm regulation of TDO by HOXA10, Ishikawa cells, HESC, and decidual cells were transfected with a HOXA10 expressing plasmid. Decidual cells were also transfected with HOXA10 antisense. Successful transfection of cells with the HOXA10-containing vector was confirmed by examination of relative Hoxa10 expression using quantitative RT-PCR. In Ishikawa cells, Hoxa10 mRNA levels were increased 120-fold (P < 0.05) in cells transfected with HOXA10-containing plasmid compared with controls. In HESC cells, the increase in HOXA10 expression was 5,853-fold (P < 0.05). In decidual cells transfected with HOXA10-containing plasmid, Hoxa10 expression increased 1100- fold (P < 0.05). Transfection of decidual cells with HOXA10 antisense-plasmid decreased Hoxa10 expression twofold (P < 0.05). Increased expression of HOXA10 resulted in alterations of TDO expression in Ishikawa Cells, HESC, and decidual cells; however, HOXA10 transfection led to differential effects on TDO expression in these cell lines. In Ishikawa cells, transfection with HOXA10 vector led to significantly lower amounts of TDO expression compared with controls (0.42-fold, P < 0.05; Fig. 1B). In HESC cells, transfection of HOXA10-containing vector led to increased TDO expression compared with controls (5.28-fold, P < 0.05; Fig. 1C). In decidual cells, transfection of HOXA10 vector also increased TDO expression compared with controls (1.89-fold, P < 0.05; Fig. 1D). There was a decrease in TDO expression in decidual cells transfected with HOXA10 antisense; however, this decrease was not statistically significant. Serotonin is expressed in response to Hoxa10 treatment in vivo. Because TDO activity regulates tryptophan availability for serotonin synthesis, we examined the localization of serotonin within the murine uteri of mice treated with HOXA10-expressing plasmid vectors. Serotonin localization was examined in mice treated with HOXA10/pcDNA3.1(+) plasmid and controls [treated with pcDNA3.1(+)]. Our cell culture data showed that increased HOXA10 in endometrial glands (modeled by Ishikawa cells) decreased TDO expression. Because lower TDO increases available tryptophan for serotonin synthesis, our model predicts that overexpression of HOXA10 in endometrial glands would increase serotonin. Serotonin levels were dramatically increased in the luminal and glandular epithelium of mice that were treated with HOXA10 vector compared with control mice. (Fig. 2). There were low levels of serotonin in the stroma of both control and Hoxa10-plasmid-treated mice. Effect of Hoxa10 regulation of serotonin on embryo development. Pregnant mice were transfected in vivo with Hoxa10-antisense. Embryo quality on day 4.5 postcoitum was examined in mice treated with in vivo gene transfection or control. There were fewer total embryos recovered in mice treated with Hoxa10 antisense compared with controls (P < 0.05). There was no significant variation in the number of embryos recovered individual animals in either treatment group. Control mice, transfected with empty pcDNA3.1(+) vector, had a significantly higher percentage of blastocysts per embryo recovered than mice treated with HOXA10 antisense cDNA/pcDNA3.1(+) transfection (83.9 vs. 7.7%, P < 0.05; Table 1; Fig. 3). Table 1. | Number of Embryos | Number of Embryos per Mouse (mean, range) | Percent Blastocysts | | |---|---|---|---| | Control | 31 | 7.8 (7–9) | 83.9% | | Antisense Hoxa10 | 13 | 4.3 (3–5) | 7.7% (P < 0.05) | Embryo quality is the percentage of embryos developing to the blastocyst stage. Control is vector alone. Mann-Whitney test was used to determine the significance of differences in embryo number. Independent sample t-test was performed to determine statistical significance of the differences in the percentage of embryos developing to blastocysts. Effect of serotonin receptor blockade on embryo development. Knowing that embryos express 5-HT1D and 5-HT7 receptors, we examined embryos from mice treated with antagonists (the 5-HT1D antagonist GR127935 and the 5-HT7 antagonist SB269970). Embryos were examined on day 4.5 postcoitum in mice treated with the 5-HT 1D receptor and 5-HT 7 receptor antagonists or control media on day 2 postcoitum. Embryo development was assessed by measuring the ability to develop to the blastocyst stage. Control mice (treated with M2 medium alone) had a significantly higher percentage of blastocysts per embryo recovered than mice treated with 5-HT 1D and 5-HT 7 receptor antagonists (75 vs. 20%, P < 0.05). There was a trend towards a decrease in the total number of embryos recovered in mice treated with serotonin receptor antagonists compared with controls. There was no difference in the number of embryos recovered from individual animals in either treatment group. (Table 2; Fig. 4). Table 2. | Number of Embryos | Number of Embryos per Mouse (mean, range) | Percent Blastocysts | | |---|---|---|---| | Control | 28 | 9.3 (7–11) | 75% | | Serotonin antagonist | 15 | 5 (4–6) | 20% (P < 0.05) | Control is M2 medium alone. Mann-Whitney test was used to determine the significance of differences in embryo number. Independent sample t-test was performed to determine statistical significance of the differences in the percentage of embryos developing to blastocysts. Induction of decidual response within the endometrium. Tryptophan degradation at the time of implantation is necessary to avoid T-cell-mediated immune rejection of the implanting embryo. TDO expression increases in HESC and decidual cells in response to HOXA10. TDO functions to catabolize tryptophan. Pseudopregnant mice transfected with TDO expressing plasmid in vivo and controls were subsequently treated with sesame oil to induce a decidual reaction. Immunohistochemistry was performed to examine localization of CD3, a marker present on mature T cells (Fig. 5A). Mice transfected with TDO plasmid showed significantly fewer mean CD3+ cells per high-power field than controls (1.4 vs. 12.1, respectively, P < 0.05).

TDO expression is regulated by HOXA10 within the uterus at the time of endometrial receptivity. Here we confirmed the results of our previous microarray, showing increased in TDO mRNA in whole uterine tissue in response to increased Hoxa10. We then used a cell culture model to identify the regulation of TDO expression in individual cell types. TDO was differentially regulated by HOXA10 in epithelial cells (Ishikawa), stromal cells (HESC), and decidual cells. In endometrial glandular cells, constitutive expression of HOXA10 led to a significant decrease in TDO expression. In endometrial stromal cells and decidual cells, elevated HOXA10 expression led to significant increases in TDO expression. Tryptophan is a necessary substrate for serotonin production, and TDO activity regulates tryptophan availability. TDO is differentially regulated by HOXA10 in epithelial and stromal cells. We have shown that increased Hoxa10 expression decreases TDO expression in endometrial epithelial cells. Decreased glandular tryptophan catabolism (mediated by TDO) increases available tryptophan for serotonin production. As predicted, constitutive expression of HOXA10 increased serotonin within the glandular and luminal epithelium in vivo. We further hypothesized that increased glandular serotonin may act to promote embryo development. We (5) have previously shown that litter size is increased in mice treated with in utero transfection of HOXA10. Serotonin receptors are expressed in murine preimplantation embryos (3, 15, 17). The importance of maternally derived serotonin has been demonstrated by the fact that mice with targeted disruption of tryptophan hydroxylase 1 produce offspring with abnormal central nervous system development, regardless of the genotype of the fetus (11). Interestingly, constitutive expression of HOXA10 in endometrial stromal cells and decidual cells led to increased TDO expression, the opposite of the effect seen in epithelial cells. Because TDO catabolizes tryptophan, we examined potential roles for stromal tryptophan catabolism in the peri-implantation period. We hypothesize that the increase in TDO within the stroma is important for immune tolerance of embryo implantation. T-cell proliferation is inhibited by tryptophan degradation. Before IDO expression by trophoblasts, TDO is expressed in the endometrium (21, 34). Tryptophan degradation is increased in the early embryonic period, occurs independent of IDO activity, and coincides with the time of greatest TDO expression (34). These findings are highly suggestive of a role for TDO in early embryonic tryptophan catabolism. Our results show that increased expression of TDO at the time of implantation significantly decreases the number of CD3+ T cells within the endometrial stroma. Similarly, targeted disruption of Hoxa10 leads to accumulation of T lymphocytes within the endometrial stroma at the time of implantation (46). The accumulation of T cells seen in Hoxa10-deficient mice may be due to decreased stromal tryptophan degradation by TDO. This suggests a link between maternal HOXA10 function and immune tolerance of implantation, which may be mediated through increased TDO-mediated tryptophan catabolism in the endometrial stroma. We found a significant reduction in embryo quality when embryos were exposed to 5-HT1D and 5-HT7 receptor antagonists. Because in utero treatment with serotonin antagonists is a novel technique, we chose to superovulate mice to optimize our chance of successful embryo recovery. Embryo viability between days 2.5 and 3.5 postcoitum is impaired in Hoxa10-deficient mice (32). The timing of embryo demise in these mice correlated with the timing of normal Hoxa10 expression in the distal oviduct and uterus. HOXA10-mediated suppression of glandular TDO activity, with a subsequent increase in glandular serotonin, has direct effects on embryo viability; loss of TDO activity likely mediates the embryo phenotype seen in Hoxa10-deficient mice. Embryo implantation is a complex phenomenon, likely dependent on multiple signaling molecules; here we identify an essential role for TDO in this process. HOX10 differentially regulates endometrial TDO expression in the endometrial epithelial and stromal cells. Glandular and stromal tryptophan degradation by TDO may enhance embryo development through both serotonin signaling and immune suppression. Our working model is demonstrated in Fig. 6. During the window of endometrial receptivity, when Hoxa10 levels peak, downregulation of TDO expression within the endometrial glands leads to decreased tryptophan catabolism. With more available tryptophan substrate, there is increased glandular serotonin. Serotonin signaling through the 5-HT1D and 5-HT7 receptors, known to be present on preimplantation embryos, improves embryo viability. We further hypothesize that the HOXA10-mediated increase in TDO expression in the endometrial stroma leads to immune tolerance of embryo implantation due to increased tryptophan catabolism, a mechanism known to inhibit T-cell proliferation. GRANTS This work was supported by NICHD Grants HD-052668 and HD-036887. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). ACKNOWLEDGMENTS We thank Elisa Jorgensen for excellent technical support and assistance. We also thank Jason Bromer, J. Ryan Martin, and Hongling Du for technical advice. HESC and primary third trimester human decidual cells were generous gifts from Dr. Charles Lockwood. Ishikawa cells were a generous gift from Dr. Richard Hochberg.

- 1. Acharya SB, Goswami NG, Debnath PK. Uterine and placental 5-HT profile in different gestational period of albino rats. Indian J Exp Biol 27: 505–509, 1989 [PubMed] [Google Scholar] - 2. Amenta F, Vega JA, Ricci A, Collier WL. Localization of 5-hydroxytryptamine-like immunoreactive cells and nerve fibers in the rat female reproductive system. Anat Rec 233: 478–484, 1992 [DOI] [PubMed] [Google Scholar] - 3. Amireault P, Dube F. Serotonin and its antidepressant-sensitive transport in mouse cumulus-oocyte complexes and early embryos. Biol Reprod 73: 358–365, 2005 [DOI] [PubMed] [Google Scholar] - 4. Baban B, Chandler P, McCool D, Marshall B, Munn DH, Mellor AL. Indoleamine 2,3-dioxygenase expression is restricted to fetal trophoblast giant cells during murine gestation and is maternal genome specific. J Reprod Immunol 61: 67–77, 2004 [DOI] [PubMed] [Google Scholar] - 5. Bagot CN, Troy PJ, Taylor HS. Alteration of maternal Hoxa10 expression by in vivo gene transfection affects implantation. Gene Ther 7: 1378–1384, 2000 [DOI] [PubMed] [Google Scholar] - 6. Benson GV, Lim H, Paria BC, Satokata I, Dey SK, Maas RL. Mechanisms of reduced fertility in Hoxa-10 mutant mice: uterine homeosis and loss of maternal Hoxa-10 expression. Development 122: 2687–2696, 1996 [DOI] [PubMed] [Google Scholar] - 7. Bodis J, Bognar Z, Hartmann G, Torok A, Csaba IF. Measurement of noradrenaline, dopamine and serotonin contents in follicular fluid of human graafian follicles after superovulation treatment. Gynecol Obstet Invest 33: 165–167, 1992 [DOI] [PubMed] [Google Scholar] - 8. Bodis J, Torok A, Tinneberg h, Hanf V, Papenfuss F, Schwarz H. Serotonin induces progesterone release from human granulosa cells in a superfused granulosa cell system. Arch Gynecol Obstet 253: 59–64, 1993 [DOI] [PubMed] [Google Scholar] - 9. Cermik D, Selam B, Taylor HS. Regulation of HOXA-10 expression by testosterone in vitro and in the endometrium of patients with polycystic ovary syndrome. J Clin Endocrinol Metab 88: 238–243, 2003 [DOI] [PubMed] [Google Scholar] - 10. Clausell DE, Soliman KF. Ovarian serotonin content in relation to ovulation. Experientia 34: 410–411, 1978 [DOI] [PubMed] [Google Scholar] - 11. Cote F, Fligny C, Bayard E, Launay JM, Gershon MD, Mallet J, Vodjdani G. Maternal serotonin is crucial for murine embryonic development. Proc Natl Acad Sci USA 104: 329–334, 2007 [DOI] [PMC free article] [PubMed] [Google Scholar] - 12. Daftary GS, Kayisli U, Seli E, Bukulmez O, Arici A, Taylor HS. Salpingectomy increases peri-implantation endometrial HOXA10 expression in women with hydrosalpinx. Fertil Steril 87: 367–372, 2007 [DOI] [PubMed] [Google Scholar] - 13. Daftary GS, Troy PJ, Bagot CN, Young SL, Taylor HS. Direct regulation of beta3-integrin subunit gene expression by HOXA10 in endometrial cells. Mol Endocrinol 16: 571–579, 2002 [DOI] [PubMed] [Google Scholar] - 14. Du H, Daftary GS, Lalwani SI, Taylor HS. Direct regulation of HOXA10 by 1,25-(OH)2D3 in human myelomonocytic cells and human endometrial stromal cells. Mol Endocrinol 19: 2222–2233, 2005 [DOI] [PubMed] [Google Scholar] - 15. Dube F, Amireault P. Local serotonergic signaling in mammalian follicles, oocytes and early embryos. Life Sci 81: 1627–1637, 2007 [DOI] [PubMed] [Google Scholar] - 16. Hsieh-Li HM, Witte DP, Weinstein M, Branford W, Li H, Small K, Potter SS. Hoxa 11 structure, extensive antisense transcription, and function in male and female fertility. Development 121: 1373–1385, 1995 [DOI] [PubMed] [Google Scholar] - 17. Il'kova G, Rehak P, Vesela J, Cikos S, Fabian D, Czikkova S, Koppel J. Serotonin localization and its functional significance during mouse preimplantation embryo development. Zygote 12: 205–213, 2004 [DOI] [PubMed] [Google Scholar] - 18. Juorio AV, Chedrese PJ, Li XM. The influence of ovarian hormones on the rat oviductal and uterine concentration of noradrenaline and 5-hydroxytryptamine. Neurochem Res 14: 821–827, 1989 [DOI] [PubMed] [Google Scholar] - 19. Krikun G, Mor G, Alvero A, Guller S, Schatz F, Sapi E, Rahman M, Caze R, Qumsiyeh M, Lockwood CJ. A novel immortalized human endometrial stromal cell line with normal progestational response. Endocrinology 145: 2291–2296, 2004 [DOI] [PubMed] [Google Scholar] - 20. Kroeze WK, Kristiansen K, Roth BL. Molecular biology of serotonin receptors structure and function at the molecular level. Curr Top Med Chem 2: 507–528, 2002 [DOI] [PubMed] [Google Scholar] - 21. Kudo Y, Hara T, Katsuki T, Toyofuku A, Katsura Y, Takikawa O, Fujii T, Ohama K. Mechanisms regulating the expression of indoleamine 2,3-dioxygenase during decidualization of human endometrium. Hum Reprod 19: 1222–1230, 2004 [DOI] [PubMed] [Google Scholar] - 22. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2[-delta delta C(T)] method. Methods 25: 402–408, 2001 [DOI] [PubMed] [Google Scholar] - 23. Lockwood CJ, Murk WK, Kayisli UA, Buchwalder LF, Huang SJ, Arcuri F, Li M, Gopinath A, Schatz F. Regulation of interleukin-6 expression in human decidual cells and its potential role in chorioamnionitis. Am J Pathol 177: 1755–1764 [DOI] [PMC free article] [PubMed] [Google Scholar] - 24. Lu Z, Hardt J, Kim JJ. Global analysis of genes regulated by HOXA10 in decidualization reveals a role in cell proliferation. Mol Hum Reprod 14: 357–366, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar] - 25. McKercher TC, Van Orden LS, 3rd, Bhatnagar RK, Burke JP. Estrogen-induced biogenic amine reduction in rat uterus. J Pharmacol Exp Ther 185: 514–522, 1973 [PubMed] [Google Scholar] - 26. Minatogawa Y, Suzuki S, Ando Y, Tone S, Takikawa O. Tryptophan pyrrole ring cleavage enzymes in placenta. Adv Exp Med Biol 527: 425–434, 2003 [DOI] [PubMed] [Google Scholar] - 27. Miura H, Ozaki N, Sawada M, Isobe K, Ohta T, Nagatsu T. A link between stress and depression: shifts in the balance between the kynurenine and serotonin pathways of tryptophan metabolism and the etiology and pathophysiology of depression. Stress 11: 198–209, 2008 [DOI] [PubMed] [Google Scholar] - 28. Munn DH, Zhou M, Attwood JT, Bondarev I, Conway SJ, Marshall B, Brown C, Mellor AL. Prevention of allogeneic fetal rejection by tryptophan catabolism. Science 281: 1191–1193, 1998 [DOI] [PubMed] [Google Scholar] - 29. Penna I, Du H, Ferriani R, Taylor HS. Calpain5 expression is decreased in endometriosis and regulated by HOXA10 in human endometrial cells. Mol Hum Reprod 14: 613–618, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar] - 30. Rackow BW, Taylor HS. Submucosal uterine leiomyomas have a global effect on molecular determinants of endometrial receptivity. Fertil Steril 93: 2027–2034, 2010 [DOI] [PMC free article] [PubMed] [Google Scholar] - 31. Salter M, Hazelwood R, Pogson CI, Iyer R, Madge DJ. The effects of a novel and selective inhibitor of tryptophan 2,3-dioxygenase on tryptophan and serotonin metabolism in the rat. Biochem Pharmacol 49: 1435–1442, 1995 [DOI] [PubMed] [Google Scholar] - 32. Satokata I, Benson G, Maas R. Sexually dimorphic sterility phenotypes in Hoxa10-deficient mice. Nature 374: 460–463, 1995 [DOI] [PubMed] [Google Scholar] - 33. Smith CC, Taylor HS. Xenoestrogen exposure imprints expression of genes (Hoxa10) required for normal uterine development. FASEB J 21: 239–246, 2007 [DOI] [PubMed] [Google Scholar] - 34. Suzuki S, Tone S, Takikawa O, Kubo T, Kohno I, Minatogawa Y. Expression of indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase in early concepti. Biochem J 355: 425–429, 2001 [DOI] [PMC free article] [PubMed] [Google Scholar] - 35. Tatsumi K, Higuchi T, Fujiwara H, Nakayama T, Egawa H, Itoh K, Fujii S, Fujita J. Induction of tryptophan 2,3-dioxygenase in the mouse endometrium during implantation. Biochem Biophys Res Commun 274: 166–170, 2000 [DOI] [PubMed] [Google Scholar] - 36. Taylor HS, Arici A, Olive D, Igarashi P. HOXA10 is expressed in response to sex steroids at the time of implantation in the human endometrium. J Clin Invest 101: 1379–1384, 1998 [DOI] [PMC free article] [PubMed] [Google Scholar] - 37. Taylor HS, Bagot C, Kardana A, Olive D, Arici A. HOX gene expression is altered in the endometrium of women with endometriosis. Hum Reprod 14: 1328–1331, 1999 [DOI] [PubMed] [Google Scholar] - 38. Taylor HS, Igarashi P, Olive DL, Arici A. Sex steroids mediate HOXA11 expression in the human peri-implantation endometrium. J Clin Endocrinol Metab 84: 1129–1135, 1999 [DOI] [PubMed] [Google Scholar] - 39. Taylor HS, Van den Heuvel GB, Igarashi P. A conserved Hox axis in the mouse and human female reproductive system: late establishment and persistent adult expression of the Hoxa cluster genes. Biol Reprod 57: 1338–1345, 1997 [DOI] [PubMed] [Google Scholar] - 40. Taylor MW, Feng GS. Relationship between interferon-gamma, indoleamine 2,3-dioxygenase, and tryptophan catabolism. FASEB J 5: 2516–2522, 1991 [PubMed] [Google Scholar] - 41. Tetrault AM, Richman SM, Fei X, Taylor HS. Decreased endometrial HOXA10 expression associated with use of the copper intrauterine device. Fertil Steril 92: 1820–1824, 2009 [DOI] [PMC free article] [PubMed] [Google Scholar] - 42. Thackray SJ, Mowat CG, Chapman SK. Exploring the mechanism of tryptophan 2,3-dioxygenase. Biochem Soc Trans 36: 1120–1123, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar] - 43. Troy PJ, Daftary GS, Bagot CN, Taylor HS. Transcriptional repression of peri-implantation EMX2 expression in mammalian reproduction by HOXA10. Mol Cell Biol 23: 1–13, 2003 [DOI] [PMC free article] [PubMed] [Google Scholar] - 44. Vesela J, Rehak P, Mihalik J, Czikkova S, Pokorny J, Koppel J. Expression of serotonin receptors in mouse oocytes and preimplantation embryos. Physiol Res 52: 223–228, 2003 [PubMed] [Google Scholar] - 45. Vitiello D, Pinard R, Taylor H. Gene expression profiling reveals putative HOXA10 downstream targets in the periimplantation mouse uterus. Reprod Sci 15: 529–535, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar] - 46. Yao MW, Lim H, Schust DJ, Choe SE, Farago A, Ding Y, Michaud S, Church GM, Maas RL. Gene expression profiling reveals progesterone-mediated cell cycle and immunoregulatory roles of Hoxa-10 in the preimplantation uterus. Mol Endocrinol 17: 610–627, 2003 [DOI] [PubMed] [Google Scholar]