Abstract
During pregnancy in several species including humans and rodents, the endometrium undergoes decidualization. This process of differentiation from endometrial to decidual tissue occurs only after the onset of implantation in mice. It can also be artificially induced causing the formation of deciduomal tissue. The purpose of this study was to compare the gene expression profile of the developing decidua in pregnant mice with the deciduoma formed after artificial induction in an effort to identify conceptus-influenced changes in uterine gene expression during decidualization. We induced decidualization artificially by transferring blastocyst-sized ConA-coated agarose beads into the uterus on day 2.5 of pseudopregnancy. Recently published work has found this model to be more ‘physiological’ than other methods. Total RNA was isolated from blastocyst and bead-induced ‘implantation’ sites of the uteri of day 7.5 pregnant (decidua) and pseudopregnant (deciduoma) mice respectively. This RNA was then used for microarray analysis using Mouse Illumina BeadArray chips. This analysis revealed potential differential mRNA levels of only 45 genes between the decidua and bead-induced deciduoma tissues. We confirmed the differential mRNA levels of 31 of these genes using quantitative RT-PCR. Finally, the level and localization of some of the mRNAs for select genes (Aldh3a1, Bcmo1, Guca2b, and Inhbb) identified by our microarray analysis were examined in more detail. This study provides the identity of a small set of genes whose expression in the uterus during decidualization may be influenced by molecular signals from the conceptus.
Introduction
In mammals, implantation begins with the interaction of the conceptus with the uterine epithelium and ends in the formation of the definitive placenta. In some species such as humans and rodents, the endometrium undergoes decidualization during implantation (reviewed in Krehbiel (1937), Tachi & Tachi (1974), Abrahamsohn & Zorn (1993), Abrahamsohn et al. (2002) and Gellersen et al. (2007)). Decidualization is the process where the endometrial tissue changes into decidual tissue and is critical in the establishment of pregnancy. The changes that occur during decidualization include differentiation of the endometrial fibroblast-like cells into epithelioid-like decidual cells, extracellular matrix remodeling, apoptosis, angiogenesis, and changes in immune cell populations. The endometrial tissue is believed to provide a key environment required for the developing conceptus until the functional placenta is formed. Although only select species are said to exhibit uterine decidualization, similar changes have been suggested for other species including those where implantation is superficial (Mossman 1937, Kellas 1966, King et al. 1980, MacIntyre et al. 2002, Johnson et al. 2003, Joyce et al. 2005). However, more work is required to characterize the similarities and differences between mammalian species with quite different types of implantation.
The decidualization response can be initiated by different means depending on species. For example, in humans, decidualization begins near the end of the secretory phase of the menstrual cycle (Gellersen et al. 2007). This is driven by the effects of progesterone from the ovary, and if pregnancy is not obtained, menstruation occurs. However, if pregnancy is obtained, decidualization continues as the human conceptus undergoes implantation. In the rodent uterus, the situation is slightly different. Although there is no doubt that it is also progesterone dependent in rodent species (Psychoyos 1973), decidualization will not begin unless there is an ‘implantation stimulus’. This stimulus is likely physical in nature (Kennedy et al. 2007). The requirement for a physical implantation stimulus in mice and rats is exemplified by many models, where decidualization is induced artificially in the uteri of pseudopregnant, pregnant, or ovariectomized hormone-sensitized animals. Methods to artificially induce decidualization have ranged from quite traumatic stimuli such as electrical shock (Loeb, 1908) to those that are believed to be more physiological such as intraluminal instillation of sesame oil (Deb et al. 2006). In all cases, the tissue that forms in response to an artificial stimulus is called a deciduoma to discern it from the decidua that forms in pregnant animals.
Previous work has suggested that the mammalian conceptus may secrete paracrine factors that can regulate endometrial gene expression during decidualization (reviewed in Herington & Bany (2009)). Clear examples of this exist in several species. For example, it is certain that human chorionic gonadotrophin and interferon τ are secreted by the human and bovine conceptuses respectively, and they regulate gene expression in the endometrium during implantation (Thatcher et al. 2001, Licht et al. 2007, Spencer et al. 2007, Sherwin et al. 2008). Recently, we and others have sought to find such conceptus factors as well as targeted endometrial genes in the mouse (Austin et al. 2003, Bany & Cross 2006, Kashiwagi et al. 2007, Herington et al. 2009). The approach used was to compare uterine gene expression between the decidua and oil-induced deciduomas. This approach seemed reasonable at the time as the oil-induced deciduoma models were the most commonly used. As a consequence of this work, a large number of genes were identified whose expression in the endometrium may be under the direct or indirect control of conceptus-derived paracrine signals. Recently, however, several different artificial models of decidualization were compared and suggest that a Concanavalin A-coated agarose bead model in pseudopregnant mice may have several advantages over the oil-induced deciduoma models (Herington et al. 2009). For example, a focal deciduogenic stimulus is provided by the bead, and the localized decidualization that occurs is similar to that of pregnancy. The deciduogenic stimulus provided by oil infusion into the lumen is along the entire length of the uterine horn. Therefore, the bead-induced decidualization model was used in this study, as it may be a more physiologically relevant. In this study, we performed a large-scale analysis to determine what genes may be differentially expressed in the uteri of pseudopregnant mice undergoing bead-induced decidualization compared with that of the pregnant uterus during decidualization using the Illumina platform.
Results
Microarray analysis
To determine the potential effect of the conceptus on uterine gene expression during decidualization, we compared the global mRNA levels between RNA samples from day 7.5 implantation site (IS) decidua and bead-induced deciduoma (BID) uterine segments using Illumina BeadChip microarray analysis. Of the 46 633 probes, signals for 13 063 were detected significantly above background in all samples. Analysis of the data suggested that 53 of these showed a significant difference in the level of hybridization signals between the two tissue types. Of these 53 probes, five (probe IDs 103390487, 100050500, 101340040, 2850731, and 2100170) could not be annotated or mapped to the mouse genome and thus were removed from further analysis. The remaining 48 represent the mRNAs for 45 different genes, of which 27 (Table 1) and 18 (Table 2) had greater hybridization signals in the BID and IS segment RNA samples respectively. A visualization of the relative level of the hybridization signals of these probes is provided in a heat map found in Fig. 1. Full annotation of the 45 genes is shown in Supplementary Table 1, see section on supplementary data given at the end of this article. Finally, the hypogeometric tests for gene ontology (GO) terms revealed significant overrepresentation of GO terms for biological processes and molecular function (see Supplementary Table 2, see section on supplementary data given at the end of this article).
Genes whose steady-state mRNA levels are significantly lower in the day 7.5 decidua than in the day 7.5 bead-induced deciduoma (BID). Probe ID, Illumina Probe identification number; Fold, average decidual compared to BID tissue levels.
| Symbol | Description | Probe ID | Fold |
|---|---|---|---|
| Ear2 | Eosinophil-associated, RNase A family, member 2 | 2360471 | 0.08 |
| Ltf | Lactotransferrin | 6770364 | 0.11 |
| Ear2 | Eosinophil-associated, RNase A family, member 2 | 5670239 | 0.12 |
| Padi1 | Peptidylarginine deiminase, type I | 5080270 | 0.12 |
| Klk1b27 | Kallikrein 1-related peptidase b27 | 5900632 | 0.17 |
| Dctn1 | Dynactin 1 | 1450056 | 0.19 |
| Klk1b5 | Kallikrein 1-related peptidase b5 | 2350528 | 0.19 |
| 1190003J15Rik | RIKEN cDNA 1190003J15 gene | 540408 | 0.19 |
| Klk1 | Kallikrein 1 | 104730373 | 0.20 |
| Klk1b26 | Kallikrein 1-related peptidase b26 | 101850092 | 0.21 |
| Inhbb | Inhibin beta-B | 2680092 | 0.21 |
| Klk1b3 | Kallikrein 1-related peptidase b3 | 630348 | 0.21 |
| Adrbk2 | Adrenergic receptor kinase, beta 2 | 102970520 | 0.22 |
| Guca2b | Guanylate cyclase activator 2b (retina) | 4200014 | 0.22 |
| Klk1b27 | Kallikrein 1-related peptidase b27 | 2810008 | 0.22 |
| 6720418B01Rik | RIKEN cDNA 6720418B01 gene | 103870341 | 0.24 |
| Klk1 | Kallikrein 1 | 4010706 | 0.24 |
| Fcgbp | Fc fragment of IgG-binding protein | 102570725 | 0.25 |
| 2310043J07Rik | RIKEN cDNA 2310043J07 gene | 102230139 | 0.25 |
| Lcn2 | Lipocalin 2 | 2510112 | 0.25 |
| Tmprss4 | Transmembrane protease, serine 4 | 1050402 | 0.25 |
| Erv3 | Endogenous retroviral sequence 3 | 3130338 | 0.25 |
| 2310033E01Rik | RIKEN cDNA 2310033E01 gene | 102630524 | 0.26 |
| 9030607L20Rik | RIKEN cDNA 9030607L20 gene | 106020102 | 0.27 |
| 2900076A13Rik | RIKEN cDNA 2900076A13 gene | 102900722 | 0.27 |
| Taok2 | TAO kinase 2 | 2810484 | 0.29 |
| F830028O17Rik | RIKEN cDNA F830028O17 gene | 102480324 | 0.29 |
| Col3a1 | Collagen, type III, alpha 1 | 6420273 | 0.30 |
| Foxp4 | Forkhead box P4 | 107050112 | 0.32 |
| Tmem14c | Transmembrane protein 14C | 3940193 | 0.34 |
Genes whose steady-state mRNA levels are significantly greater in the day 7.5 decidua than in the day 7.5 bead-induced deciduoma (BID). Probe ID, Illumina Probe identification number; Fold, average decidual compared to BID tissue levels.
| Symbol | Description | Probe ID | Fold |
|---|---|---|---|
| Capn6 | Calpain 6 | 1740168 | 2.54 |
| Hist1h2ah | Histone cluster 1, H2ah | 3710333 | 2.89 |
| Cald1 | Caldesmon 1 | 103870092 | 2.96 |
| Crybg3 | Beta-gamma crystallin domain containing 3 | 1770048 | 3.00 |
| Cpd | Carboxypeptidase D | 5860685 | 3.01 |
| St8sia4 | ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 4 | 3060215 | 3.06 |
| Pik3r3 | Phosphatidylinositol 3 kinase, regulatory subunit, polypeptide 3 (p55) | 100130097 | 3.10 |
| Ankrd17 | Ankyrin repeat domain 17 | 2120402 | 3.14 |
| Pfdn4 | Prefoldin 4 | 4010286 | 3.37 |
| Zfp800 | Zinc finger protein 800 | 2350253 | 3.37 |
| Mtap7d2 | MAP7 domain containing 2 | 130471 | 3.51 |
| Stmn2 | Stathmin-like 2 | 6220181 | 3.57 |
| Aldh3a1 | Aldehyde dehydrogenase family 3, subfamily A1 | 580095 | 3.58 |
| Usmg5 | Upregulated during skeletal muscle growth 5 | 3710181 | 4.27 |
| Bcmo1 | Beta-carotene 15,15′-monooxygenase | 4780121 | 4.94 |
| Cst9 | Cystatin 9 | 2230706 | 6.73 |
| Prps2 | Phosphoribosyl pyrophosphate synthetase 2 | 3140707 | 7.20 |
| 2900060B14Rik | RIKEN cDNA 2900060B14 gene | 102810039 | 9.59 |
Heat map of differentially expressed genes between the day 7.5 decidua and deciduoma (BID). Red to black to blue indicates a gradient of high to low expression.
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
Heat map of differentially expressed genes between the day 7.5 decidua and deciduoma (BID). Red to black to blue indicates a gradient of high to low expression.
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
Heat map of differentially expressed genes between the day 7.5 decidua and deciduoma (BID). Red to black to blue indicates a gradient of high to low expression.
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
As previously shown (Bany & Cross 2006, Herington et al. 2009), hybridization signals of several markers of decidualization including Bmp2, Bmp8a, Fkbp5, and Hand2 were not significantly different between the IS and BID segments. Furthermore, since they are the cells of the conceptus in most intimate contact with the endometrium, it is also notable that hybridization signals of several trophoblast cell-specific mRNAs (Simmons et al. 2008) including Tpbpa, Tpbpb, several prolactin-related genes (Prl2a1, Prl3b1, Prl5a1, Prl7a1, Prl7a2, Prl7c1, Prl7d1, and Prl8a1), and Hand1 were not detected above background. This suggests that the samples used for the microarray analysis had negligible levels of contaminating trophoblast and ectoplacental cone tissues. Notably, although originally believed to be trophoblast specific and expressed only later in pregnancy (Hwang et al. 2000), Prl8a9 mRNA was detected in all microarray samples in this study. This confirms the recent work (Simmons et al. 2008) showing that it is expressed at low levels in the maternal decidual cells on day 7.5.
Independent verification of microarray results
Of the 27 genes examined further, we verified differential expression of 23 between the day 7.5 IS and BID segment tissues using quantitative real-time PCR (qRT-PCR; Table 3). The steady-state mRNA levels of 8 of these 23 genes were significantly greater in the IS segment tissue, while that of the remaining 15 were significantly greater in the BID segment tissue. The expression of four additional genes identified as differentially expressed in the microarray analysis was also studied in more detail as described below.
qRT-PCR verification of differential expression between the day 7.5 implantation (IS) and day 7.5 pseudopregnant bead-induced deciduoma (BID) segments.
| Symbol | IS | BID | P value |
|---|---|---|---|
| 11190003J15Rik | 1.0±0.12 | 7.0±1.44 | 0.002 |
| 2310033E01Rik | 1.0±0.20 | 10.2±2.69 | 0.001 |
| 2310043J07Rik | 1.0±0.20 | 3.5±1.21 | 0.034 |
| 2900060B14Rik | 1.0±0.11 | 0.5±0.05 | 0.003 |
| 9030607L20Rik | 1.0±0.17 | 7.9±2.74 | 0.013 |
| Adrbk2 | 1.0±0.28 | 11.6±3.61 | 0.003 |
| Ankrd17 | 1.0±0.20 | 1.4±0.36 | 0.293* |
| Cald1 | 1.0±0.18 | 0.3±0.07 | 0.008 |
| Col3a1 | 1.0±0.17 | 2.3±0.32 | 0.003 |
| Cpd | 1.0±0.27 | 0.07±0.02 | 0.014 |
| Crybg3 | 1.0±0.15 | 0.35±0.07 | 0.004 |
| Dctn1 | 1.0±0.25 | 1.7±0.24 | 0.081* |
| Ear2 | 1.0±0.33 | 166.5±20.00 | 0.000 |
| Erv3 | 1.0±0.27 | 1.5±0.19 | 0.122* |
| Foxp4 | 1.0±0.24 | 1.5±0.18 | 0.114* |
| Klk1 | 1.0±0.18 | 10.2±3.13 | 0.004 |
| Klk1b27 | 1.0±0.21 | 42.5±8.8 | 0.000 |
| Lcn2 | 1.0±0.09 | 7.4±1.92 | 0.01 |
| Ltf | 1.0±0.08 | 12.8±3.45 | 0.002 |
| Padi1 | 1.0±0.05 | 12.8±2.1 | 0.003 |
| Pfdn4 | 1.0±0.13 | 0.35±0.13 | 0.006 |
| Prps2 | 1.0±0.11 | 0.3±0.08 | 0.009 |
| Stmn2 | 1.0±0.10 | 0.5±0.18 | 0.041 |
| Taok2 | 1.0±0.18 | 14.7±4.84 | 0.002 |
| Tmem14c | 1.0±0.15 | 2.2±0.60 | 0.043 |
| Tmprss4 | 1.0±0.15 | 6.7±1.11 | 0.000 |
| Zfp800 | 1.0±0.17 | 0.24±0.06 | 0.002 |
Values represent the mean (±s.e.m.; N=4–6) relative mRNA levels normalized to 18S rRNA levels. *Not significantly different.
Expression of select genes involved in retinoic acid homeostasis
The beta-carotene 15,15′-monooxygenase (Bcmo1) gene encodes a protein (BCMO1), which catalyzes the conversion of β-carotene to retinal and is important for the production of retinoic acid (Barua & Olson 2000, Hessel et al. 2007; Fig. 2A). Since this gene appeared to be differentially expressed on day 7.5 in our microarray analysis, we further examined uterine expression during decidualization of this and related genes involved in retinoic acid metabolism. Initially, we verified significant (P<0.01) differential expression by qRT-PCR (Fig. 2B). Next, we examined Bcmo1 expression in the pregnant mouse uterus on days 3.5–8.5 (Fig. 2C). Bcmo1 mRNA levels were low in the uterus on days 3.5–4.5 of pregnancy. However, the levels significantly (P<0.01) increased on days 5.5–8.5 in IS segment compared with non-implantation site (NIS) segment. To further define the expression differences during decidualization, we determined the levels of Bcmo1 mRNA in IS segment compared to BID segment of the uterus on days 4.5–8.5 of pregnancy and pseudopregnancy respectively (Fig. 2D). The mRNA levels were significantly (P<0.01) greater in the IS segment than in the BID segment only on day 7.5.
qRT-PCR analysis of steady-state mRNA levels of select genes involved in retinoic acid metabolism. (A) Simplified schematic of the retinoic acid homeostasis pathways and the genes whose steady-state mRNA levels we examined. (B) Relative Bcmo1 mRNA levels in day 7.5 bead-induced deciduoma (BID) segment compared with implantation site (IS) segment tissues. (C) Relative Bcmo1 mRNA levels in the non-implantation site (NIS) segment compared with the IS segment on days 3.5–8.5 of pregnancy. (D) Relative Bcmo1 mRNA levels in the BID segment compared with the IS segment on days 3.5–8.5 of pseudopregnancy and pregnancy respectively. Relative levels of (E) Aldh1a3, (F) Rdh10, (G) Rdh11, and (H) Rdh14 between the BID and IS segments on day 7.5. (I) Relative Cyb26b1 mRNA levels in the BID segment compared with the IS segment on day 7.5 of pseudopregnancy and pregnancy respectively. (J) Relative Cyb26b1 mRNA levels in the NIS and IS segments on days 3.5–8.5 of pregnancy respectively. *P<0.05 and **P<0.01. Bars represent mean±s.e.m. (N=4).
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
qRT-PCR analysis of steady-state mRNA levels of select genes involved in retinoic acid metabolism. (A) Simplified schematic of the retinoic acid homeostasis pathways and the genes whose steady-state mRNA levels we examined. (B) Relative Bcmo1 mRNA levels in day 7.5 bead-induced deciduoma (BID) segment compared with implantation site (IS) segment tissues. (C) Relative Bcmo1 mRNA levels in the non-implantation site (NIS) segment compared with the IS segment on days 3.5–8.5 of pregnancy. (D) Relative Bcmo1 mRNA levels in the BID segment compared with the IS segment on days 3.5–8.5 of pseudopregnancy and pregnancy respectively. Relative levels of (E) Aldh1a3, (F) Rdh10, (G) Rdh11, and (H) Rdh14 between the BID and IS segments on day 7.5. (I) Relative Cyb26b1 mRNA levels in the BID segment compared with the IS segment on day 7.5 of pseudopregnancy and pregnancy respectively. (J) Relative Cyb26b1 mRNA levels in the NIS and IS segments on days 3.5–8.5 of pregnancy respectively. *P<0.05 and **P<0.01. Bars represent mean±s.e.m. (N=4).
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
qRT-PCR analysis of steady-state mRNA levels of select genes involved in retinoic acid metabolism. (A) Simplified schematic of the retinoic acid homeostasis pathways and the genes whose steady-state mRNA levels we examined. (B) Relative Bcmo1 mRNA levels in day 7.5 bead-induced deciduoma (BID) segment compared with implantation site (IS) segment tissues. (C) Relative Bcmo1 mRNA levels in the non-implantation site (NIS) segment compared with the IS segment on days 3.5–8.5 of pregnancy. (D) Relative Bcmo1 mRNA levels in the BID segment compared with the IS segment on days 3.5–8.5 of pseudopregnancy and pregnancy respectively. Relative levels of (E) Aldh1a3, (F) Rdh10, (G) Rdh11, and (H) Rdh14 between the BID and IS segments on day 7.5. (I) Relative Cyb26b1 mRNA levels in the BID segment compared with the IS segment on day 7.5 of pseudopregnancy and pregnancy respectively. (J) Relative Cyb26b1 mRNA levels in the NIS and IS segments on days 3.5–8.5 of pregnancy respectively. *P<0.05 and **P<0.01. Bars represent mean±s.e.m. (N=4).
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
To determine the localization of expression, we attempted to localize Bcmo1 mRNA in the uterus on days 5.5–8.5 of pregnancy or pseudopregnancy by in situ hybridization. No hybridization signals were detected with sense probes in any of the tissues as well as with antisense probes in uterine tissues from day 5.5 and in NIS or non-bead (NB) segments on days 6.5–8.5 (data not shown). On day 6.5, for both IS (Fig. 3A and B) and BID (Fig. 3C and D) segments, Bcmo1 mRNA was localized to a small area lateral to the implanting conceptus and bead respectively. On day 7.5, the signals spread mesometrially to include maternal tissue surrounding the blood islands that form in the lateral mesometrial area in the IS (Fig. 3E and F). On the other hand, only the lateral staining was observed in the day 7.5 BID segments (Fig. 3G and H). Finally, on day 8.5, Bcmo1 mRNA signals were detected in the maternal tissue completely surrounding the conceptus (Fig. 3I and J). However, hybridization signals were mainly detected in the maternal tissue lateral to the bead stimulus in the BID segments on day 8.5 of pseudopregnancy (Fig. 3K and L).
Localization of Bcmo1 mRNA in implantation site (IS) and bead-induced deciduoma (BID) segments of pregnant and pseudopregnant mice. Photomicrographs of day 6.5 IS (A and B) or BID (C and D), day 7.5 IS (E and F) or BID (G and H), and day 8.5 IS (I and J) or BID (K and L) segments are shown. AM, anti-mesometrial side; C, conceptus; IS, implantation site; MM, mesometrial side. Numbers above scale bars are in microns. Representative of at least three independent samples. Global linear adjustments of the brightness and color level were made on the photomicrographs to more accurately represent what was observed on the slides under the microscope.
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
Localization of Bcmo1 mRNA in implantation site (IS) and bead-induced deciduoma (BID) segments of pregnant and pseudopregnant mice. Photomicrographs of day 6.5 IS (A and B) or BID (C and D), day 7.5 IS (E and F) or BID (G and H), and day 8.5 IS (I and J) or BID (K and L) segments are shown. AM, anti-mesometrial side; C, conceptus; IS, implantation site; MM, mesometrial side. Numbers above scale bars are in microns. Representative of at least three independent samples. Global linear adjustments of the brightness and color level were made on the photomicrographs to more accurately represent what was observed on the slides under the microscope.
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
Localization of Bcmo1 mRNA in implantation site (IS) and bead-induced deciduoma (BID) segments of pregnant and pseudopregnant mice. Photomicrographs of day 6.5 IS (A and B) or BID (C and D), day 7.5 IS (E and F) or BID (G and H), and day 8.5 IS (I and J) or BID (K and L) segments are shown. AM, anti-mesometrial side; C, conceptus; IS, implantation site; MM, mesometrial side. Numbers above scale bars are in microns. Representative of at least three independent samples. Global linear adjustments of the brightness and color level were made on the photomicrographs to more accurately represent what was observed on the slides under the microscope.
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
The microarray data indicated that several genes involved in retinoic acid homeostasis and action were expressed at similar levels in the day 7.5 IS and BID tissues. To verify this and determine the occurrence of false-negative microarray findings, we independently measured expression levels using qRT-PCR for a small number of these targets. The aldehyde dehydrogenase 1a3 gene (Aldh1a3) encodes a protein that plays a critical role of converting retinal to retinoic acid (Fig. 2A). As shown in Fig. 2E, we confirmed that Aldh1a3 mRNA levels were not significantly (P>0.05) different between the IS and BID segments on day 7.5. The Rdh10 gene encodes a protein that can oxidize retinol into retinal and thus is important in retinoic acid homeostasis (Fig. 2A). As shown in Fig. 2F, we confirmed that retinol dehydrogenase 10 (Rdh10) mRNA levels were not significantly (P>0.05) different between the IS and BID segments on day 7.5. The retinol dehydrogenase 11 (Rdh11, previously called Psdr1) and 14 (Rdh14, previously called Pan2) genes encode proteins that can reduce retinal into retinol (Fig. 2A). We concluded that Rdh11 (Fig. 2G) plus Rdh14 (Fig. 2H) mRNA levels were similar between the IS and BID segments on day 7.5. Finally, the cytochrome P450 family 26 subfamily b polypeptide 1 (Cyp26b1) gene encodes a protein that degrades retinoic acid in tissues (White et al. 2000). We confirmed that mRNA levels did not differ between the IS and BID segments on day 7.5 (Fig. 2I). Interestingly, Cyp26b1 mRNA levels were significantly greater in the IS segment than in the NIS segment of pregnant uteri on days 7.5 and 8.5 (Fig. 2J).
Expression of type 3 aldehyde dehydrogenases
The type 3a1 aldehyde dehydrogenase gene (Aldh3a1) encodes a protein (ALDH3A1) that is believed to be involved in the breakdown of products of medium-chained lipid peroxidation in the eye (Pappa et al. 2003). Since this gene appeared to be differentially expressed on day 7.5 in our microarray analysis, we further examined its expression in the uterus during decidualization. We confirmed that Aldh3a1 mRNA levels were significantly greater (P<0.05) in the IS segment than in the BID segment on day 7.5 by qRT-PCR (Fig. 4A). In addition, we examined the expression in the pregnant mouse uterus on days 3.5–8.5 (Fig. 4B). The mRNA levels dramatically increased and were significantly greater (P<0.05) in the IS segment than in the NIS segment only on days 7.5 and 8.5. Aldh3a1 mRNA was localized in uterine sections from day 5.5 to 8.5 pregnant or pseudopregnant mice by in situ hybridization (Fig. 5). Hybridization signals were never detected in uterine tissues on day 5.5 and in NIS or NB segments on days 6.5–8.5 (data not shown). On day 6.5, for both IS (Fig. 5A and B) and BID (Fig. 5C and D) segments, Aldh3a1 mRNA was localized to a small area just lateral to the implanting conceptus or bead. On day 7.5, the signals spread mesometrially to include maternal tissue surrounding the blood islands that form in the lateral mesometrial area in the IS (Fig. 5E and F) and BID (Fig. 5G and H) segments. Finally, on day 8.5, Aldh3a1 mRNA signals were detected in the maternal tissue completely surrounding the conceptus (Fig. 5I and J). However, in the BID segments of the pseudopregnant mice, hybridization signals were only detected laterally in the mesometrial–anti-mesometrial border of the deciduoma tissue (Fig. 5K and L).
qRT-PCR analysis of steady-state type 3 aldehyde dehydrogenase mRNA levels in the mouse uterus during decidualization. (A) Aldh3a1 mRNA levels in bead-induced deciduoma (BID) segment compared with implantation site (IS) segment on day 7.5. (B) Aldh3a1 mRNA levels on days 3.5–8.5 of pregnancy in the non-implantation site (NIS) segment compared with the IS segment. (C) Aldh3a2 mRNA levels in the BID segment compared with the IS segment on day 7.5. (D) Aldh3a2 mRNA levels on days 3.5–8.5 of pregnancy in the NIS segment compared with the IS segment. *P<0.05. Bars represent mean±s.e.m. (N=4–5).
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
qRT-PCR analysis of steady-state type 3 aldehyde dehydrogenase mRNA levels in the mouse uterus during decidualization. (A) Aldh3a1 mRNA levels in bead-induced deciduoma (BID) segment compared with implantation site (IS) segment on day 7.5. (B) Aldh3a1 mRNA levels on days 3.5–8.5 of pregnancy in the non-implantation site (NIS) segment compared with the IS segment. (C) Aldh3a2 mRNA levels in the BID segment compared with the IS segment on day 7.5. (D) Aldh3a2 mRNA levels on days 3.5–8.5 of pregnancy in the NIS segment compared with the IS segment. *P<0.05. Bars represent mean±s.e.m. (N=4–5).
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
qRT-PCR analysis of steady-state type 3 aldehyde dehydrogenase mRNA levels in the mouse uterus during decidualization. (A) Aldh3a1 mRNA levels in bead-induced deciduoma (BID) segment compared with implantation site (IS) segment on day 7.5. (B) Aldh3a1 mRNA levels on days 3.5–8.5 of pregnancy in the non-implantation site (NIS) segment compared with the IS segment. (C) Aldh3a2 mRNA levels in the BID segment compared with the IS segment on day 7.5. (D) Aldh3a2 mRNA levels on days 3.5–8.5 of pregnancy in the NIS segment compared with the IS segment. *P<0.05. Bars represent mean±s.e.m. (N=4–5).
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
Localization of Aldh3a1 mRNA in implantation site (IS) and bead-induced deciduoma (BID) segments of pregnant and pseudopregnant mice. Photomicrographs of day 6.5 IS (A and B) or BID (C and D), day 7.5 IS (E and F) or BID (G and H), and day 8.5 IS (I and J) or BID (K and L) segments are shown. AM, anti-mesometrial side; C, conceptus; MM, mesometrial side. Numbers above scale bars are in microns. Representative of at least three independent samples. Global linear adjustments of the brightness and color level were made on the photomicrographs to more accurately represent what was observed on the slides under the microscope.
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
Localization of Aldh3a1 mRNA in implantation site (IS) and bead-induced deciduoma (BID) segments of pregnant and pseudopregnant mice. Photomicrographs of day 6.5 IS (A and B) or BID (C and D), day 7.5 IS (E and F) or BID (G and H), and day 8.5 IS (I and J) or BID (K and L) segments are shown. AM, anti-mesometrial side; C, conceptus; MM, mesometrial side. Numbers above scale bars are in microns. Representative of at least three independent samples. Global linear adjustments of the brightness and color level were made on the photomicrographs to more accurately represent what was observed on the slides under the microscope.
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
Localization of Aldh3a1 mRNA in implantation site (IS) and bead-induced deciduoma (BID) segments of pregnant and pseudopregnant mice. Photomicrographs of day 6.5 IS (A and B) or BID (C and D), day 7.5 IS (E and F) or BID (G and H), and day 8.5 IS (I and J) or BID (K and L) segments are shown. AM, anti-mesometrial side; C, conceptus; MM, mesometrial side. Numbers above scale bars are in microns. Representative of at least three independent samples. Global linear adjustments of the brightness and color level were made on the photomicrographs to more accurately represent what was observed on the slides under the microscope.
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
The type 3a2 aldehyde dehydrogenase gene (Aldh3a2) is closely related to Aldh3a1 and is involved in oxidation of long- to medium-chained aliphatic aldehydes in the liver and thus may be involved in the detoxification of aldehydes produced by lipid peroxidation (Mitchell & Petersen 1989). The levels of Aldh3a2 mRNA did not differ between the IS and BID segment tissues on day 7.5 of pregnancy and pseudopregnancy respectively (Fig. 4C). Furthermore, Aldh3a2 mRNA levels did not differ between the NIS and IS segments except for day 7.5, where levels were significantly (P<0.05) higher in the IS segment than in the NIS segment (Fig. 4C).
Inhibin βb gene expression
Members of the inhibin gene family encode proteins that make up subunits of inhibins and activins that play several roles in reproductive endocrinology (Ethier & Findlay 2001). We confirmed that inhibin βb (Inhbb) mRNA levels were significantly greater in the BID segment than in the IS segment by qRT-PCR (Fig. 6A). To further characterize Inhbb expression in the uterus during decidualization, we next examined mRNA levels in the pregnant uterus during the peri-implantation period. Inhbb mRNA was detected at low levels in the uterus on day 3.5 of pregnancy prior to the onset of implantation (Fig. 6B). The levels did not significantly (P>0.05) change in NIS segments on days 4.5–8.5. However, on each of these days, there were significant (P<0.05) increases in Inhbb mRNA levels in the IS segment compared with those in the NIS segment of the pregnant uterus. Finally, to determine the localization of Inhbb expression in the IS and BID tissues, we used in situ hybridization (Fig. 6C–F). Hybridization signals were detected only in the vascular endothelial cells lining the blood islands in the mesometrial tissue adjacent to the implanting conceptus or bead. Notably, two different hybridization probe synthesis templates were generated using the primers shown in Supplementary Table 3B, see section on supplementary data given at the end of this article. These two probes cover different regions of the Inhbb mRNA, and localization of hybridization signals using the two different antisense probes was identical (data not shown).
Inhbb and Fst mRNA in the mouse uterus during decidualization. (A) Inhbb mRNA levels in bead-induced deciduoma (BID) segment compared with implantation site (IS) segments on day 7.5. (B) Inhbb mRNA levels on days 3.5–8.5 of pregnancy in the non-implantation site (NIS) segment compared with the IS segment. Localization of Inhbb mRNA in the IS and BID tissues of day 7.5 pregnant (C and D) and pseudopregnant (E and F) mice uteri during decidualization on day 7.5. Numbers above scale bars are in microns. In situ hybridization is representative of at least three independent samples. Global linear adjustments of the brightness and color level were made on the photomicrographs to more accurately represent what was observed on the slides under the microscope. (G) Fst mRNA levels in the BID segment compared with the IS segment on day 7.5. (H) Fst mRNA levels on days 3.5–8.5 of pregnancy in the NIS segment compared with the IS segment. *P<0.05, **P<0.01, and ***P<0.005. Bars represent mean±s.e.m. (N=4–5). AM, anti-mesometrial side; C, conceptus; MM, mesometrial side.
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
Inhbb and Fst mRNA in the mouse uterus during decidualization. (A) Inhbb mRNA levels in bead-induced deciduoma (BID) segment compared with implantation site (IS) segments on day 7.5. (B) Inhbb mRNA levels on days 3.5–8.5 of pregnancy in the non-implantation site (NIS) segment compared with the IS segment. Localization of Inhbb mRNA in the IS and BID tissues of day 7.5 pregnant (C and D) and pseudopregnant (E and F) mice uteri during decidualization on day 7.5. Numbers above scale bars are in microns. In situ hybridization is representative of at least three independent samples. Global linear adjustments of the brightness and color level were made on the photomicrographs to more accurately represent what was observed on the slides under the microscope. (G) Fst mRNA levels in the BID segment compared with the IS segment on day 7.5. (H) Fst mRNA levels on days 3.5–8.5 of pregnancy in the NIS segment compared with the IS segment. *P<0.05, **P<0.01, and ***P<0.005. Bars represent mean±s.e.m. (N=4–5). AM, anti-mesometrial side; C, conceptus; MM, mesometrial side.
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
Inhbb and Fst mRNA in the mouse uterus during decidualization. (A) Inhbb mRNA levels in bead-induced deciduoma (BID) segment compared with implantation site (IS) segments on day 7.5. (B) Inhbb mRNA levels on days 3.5–8.5 of pregnancy in the non-implantation site (NIS) segment compared with the IS segment. Localization of Inhbb mRNA in the IS and BID tissues of day 7.5 pregnant (C and D) and pseudopregnant (E and F) mice uteri during decidualization on day 7.5. Numbers above scale bars are in microns. In situ hybridization is representative of at least three independent samples. Global linear adjustments of the brightness and color level were made on the photomicrographs to more accurately represent what was observed on the slides under the microscope. (G) Fst mRNA levels in the BID segment compared with the IS segment on day 7.5. (H) Fst mRNA levels on days 3.5–8.5 of pregnancy in the NIS segment compared with the IS segment. *P<0.05, **P<0.01, and ***P<0.005. Bars represent mean±s.e.m. (N=4–5). AM, anti-mesometrial side; C, conceptus; MM, mesometrial side.
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
Follistatin (encoded by the Fst gene) binds to activins preventing their ability to activate target receptors (Ethier & Findlay 2001). Therefore, we examined the expression of the Fst gene more closely. Unlike Inhbb, microarray analysis indicates no difference in Fst and Inhba mRNA levels between the IS and BID segments on day 7.5. We confirmed that Fst mRNA levels did not differ significantly (P>0.05) between the BID and IS segments on day 7.5 of pregnancy by qRT-PCR (Fig. 6G). Further analysis of Fst mRNA levels in pregnant uteri showed that levels are significantly greater (P<0.01) in the IS segment than in the NIS segment on days 4.5–8.5 of pregnancy (Fig. 6H).
Characterization of guanylate cyclase activator 2 gene (Guca2a and Guca2b) expression
Guca2a and Guca2b genes encode secreted endocrine/paracrine hormones that bind to the same guanylate cyclase receptor and have well-characterized roles in controlling salt handling and fluid balance in the intestines and kidney (Sindic & Schlatter 2006a, 2006b, 2007). Microarray analysis indicated that Guca2b, but not Guca2a, mRNA levels differed between the BID and IS segments on day 7.5 of pseudopregnancy and pregnancy respectively. This was verified by qRT-PCR as shown in Fig. 7A and B where it was observed that the steady-state Guca2b mRNA levels were significantly greater (by ∼4.5-fold, P<0.001) in the BID segment than in the IS segment tissues. Closer examination of pregnant uteri during the peri-implantation period revealed that Guca2b mRNA levels were low on day 3.5 of pregnancy (prior to the onset of implantation) compared with the days following the onset of implantation (Fig. 7C). After the onset of implantation, Guca2b mRNA levels between the NIS segments were significantly (P<0.05) greater than that of the IS segments only on day 8.5 and no other day examined. Finally, Guca2b mRNA levels in BID segment compared to IS segment on days 3.5–8.5 of pseudopregnancy and pregnancy are shown in Fig. 7D. On day 3.5, Guca2b mRNA levels were significantly (P<0.001) greater in the pseudopregnant mice than in pregnant mice by ∼11.5-fold. On days 4.5–6.5, mRNA levels were not significantly different between the BID and IS segment tissues. Again, however, mRNA levels were significantly (P<0.05) greater in the BID segment than in IS segment tissues on day 7.5 by fivefold. On day 8.5, this difference became approximately twofold (P<0.01).
qRT-PCR analysis of Guca2b and Guca2a mRNA levels in the mouse uterus during decidualization. (A) Guca2b and (B) Guca2a mRNA levels in the bead-induced deciduoma (BID) segment compared with the implantation site (IS) segment on day 7.5. (C) Guca2b mRNA levels on days 3.5–8.5 of pregnancy in the non-implantation site (NIS) segment compared with the IS segment. (D) Guca2b mRNA levels in the BID segment compared with the IS segment on days 3.5–8.5 of pseudopregnancy or pregnancy respectively. Bars represent mean±s.e.m. (N=4). *P<0.05,**P<0.01, and ***P<0.005.
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
qRT-PCR analysis of Guca2b and Guca2a mRNA levels in the mouse uterus during decidualization. (A) Guca2b and (B) Guca2a mRNA levels in the bead-induced deciduoma (BID) segment compared with the implantation site (IS) segment on day 7.5. (C) Guca2b mRNA levels on days 3.5–8.5 of pregnancy in the non-implantation site (NIS) segment compared with the IS segment. (D) Guca2b mRNA levels in the BID segment compared with the IS segment on days 3.5–8.5 of pseudopregnancy or pregnancy respectively. Bars represent mean±s.e.m. (N=4). *P<0.05,**P<0.01, and ***P<0.005.
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
qRT-PCR analysis of Guca2b and Guca2a mRNA levels in the mouse uterus during decidualization. (A) Guca2b and (B) Guca2a mRNA levels in the bead-induced deciduoma (BID) segment compared with the implantation site (IS) segment on day 7.5. (C) Guca2b mRNA levels on days 3.5–8.5 of pregnancy in the non-implantation site (NIS) segment compared with the IS segment. (D) Guca2b mRNA levels in the BID segment compared with the IS segment on days 3.5–8.5 of pseudopregnancy or pregnancy respectively. Bars represent mean±s.e.m. (N=4). *P<0.05,**P<0.01, and ***P<0.005.
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
Assessing the potential role of giant cells
To determine the potential role of trophoblast giant cells in the differential expression of four of the genes found differentially expressed between the BID and IS segments by microarray and qRT-PCR analyses, we used mice carrying a null mutation in the Hand1 gene. Hand1-deficient conceptuses do not have trophoblast giant cells, and they die around days 7.5–8.5 (Riley et al. 1998). As shown in Table 4, Aldh3a1, Bcmo1, Guca2b, and Inhbb mRNA levels did not differ in the IS segment tissue containing Hand1 wild-type compared with those containing knockout conceptuses.
qRT-PCR to assess mRNA levels between the day 7.5 implantation (IS) segment tissues containing Hand1 wild-type (WT) and knockout (KO) conceptuses.
| Symbol | WT | KO | P value |
|---|---|---|---|
| Aldh3a1 | 1.55±0.56 | 2.35±0.90 | 0.251 |
| Bcmo1 | 1.00±0.11 | 1.38±0.56 | 0.48 |
| Guca2b | 1.00±0.30 | 1.30±0.75 | 0.56 |
| Inhbb | 1.00±0.12 | 0.78±0.05 | 0.36 |
Values represent the mean (±s.e.m.; N=5) relative mRNA levels normalized to 18S rRNA levels.
Assessing select genes previously found to be differentially expressed between the decidua and oil-induced deciduoma
Previous work indicated a great number of genes differentially expressed between the decidua and oil-induced deciduoma on day 7.5 of pregnancy and pseudopregnancy respectively (Kashiwagi et al. 2007). If the microarray data of this study indeed have a high false-negative rate, then many of the genes identified in the previously published microarray work may also be differentially expressed between the decidua and BID. Therefore, we assessed the differential expression of four additional random genes between the IS and BID tissues previously identified to be differentially expressed by Kashiwagi et al. (2007) between the decidua and oil-induced deciduoma. As shown in Table 5, Dact1, Hoxb6, Nkg7, and Prss28 (Isp1-pending in Kashiwagi et al. (2007)) mRNA levels did not differ between the BID and IS tissues on day 7.5 of pseudopregnancy and pregnancy respectively. Since Prss28 was previously shown to be one of the genes more differentially expressed between the decidua and oil-induced deciduoma, we localized its expression using in situ hybridization. Prss28 hybridization signals were detected in the glandular epithelial cells throughout day 7.5 pregnant uterus in the NIS (Fig. 8A) and IS (Fig. 8B and C) tissues. Similarly, Prss28 hybridization signals were detected in the glandular epithelial cells throughout day 7.5 pseudopregnant uterus in the interbead (Fig. 8D) and BID (Fig. 8E and F) tissues. In both the pregnant and pseudopregnant uteri, more hybridization signal was detected in the areas of the uterus not undergoing decidualization due to the higher abundance of glandular epithelial cells. To assess this further, we measured the relative mRNA levels of all four genes between the NIS and IS tissues on day 7.5 of pregnancy. As shown in Table 6, the mRNA levels of Hoxb6 and Prss28 were significantly greater in the NIS tissue than in the IS tissue. On the other hand, the mRNA levels of Dact1 did not differ significantly between these tissue types. Finally, the mRNA levels of Nkg7 were significantly greater in the IS tissue than in the NIS tissue.
qRT-PCR to assess mRNA levels between the day 7.5 implantation (IS) and bead-induced deciduoma (BID) segment tissues of pregnant and pseudopregnant mice respectively.
| Symbol | BID | IS | P value |
|---|---|---|---|
| Dact1 | 1.5±0.30 | 1.0±0.25 | >0.05 |
| Hoxb6 | 2.6±0.83 | 1.0±0.30 | >0.05 |
| Nkg7 | 1.7±0.88 | 1.0±0.23 | >0.05 |
| Prss28 | 1.8±0.40 | 1.0±0.13 | >0.05 |
Values represent the mean (±s.e.m.; N=4) relative mRNA levels normalized to 18S rRNA levels.
Localization of Prss28 mRNA in the mouse uterus on day 7.5 of pregnancy or pseudopregnancy. Representative sections from (A) non-implantation (NIS) and (B and C) implantation site (IS) segments from pregnant uteri. Representative sections from (D) non-bead (NB) and (E and F) bead-induced deciduoma (BID) segments of pseudopregnant uteri. AM, anti-mesometrial side; B, bead stimulus; C, conceptus; MM, mesometrial side. Numbers above scale bars are in microns. Sections are representative of at least three independent samples. Global linear adjustments of the brightness and color level were made on the photomicrographs to more accurately represent what was observed on the slides under the microscope.
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
Localization of Prss28 mRNA in the mouse uterus on day 7.5 of pregnancy or pseudopregnancy. Representative sections from (A) non-implantation (NIS) and (B and C) implantation site (IS) segments from pregnant uteri. Representative sections from (D) non-bead (NB) and (E and F) bead-induced deciduoma (BID) segments of pseudopregnant uteri. AM, anti-mesometrial side; B, bead stimulus; C, conceptus; MM, mesometrial side. Numbers above scale bars are in microns. Sections are representative of at least three independent samples. Global linear adjustments of the brightness and color level were made on the photomicrographs to more accurately represent what was observed on the slides under the microscope.
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
Localization of Prss28 mRNA in the mouse uterus on day 7.5 of pregnancy or pseudopregnancy. Representative sections from (A) non-implantation (NIS) and (B and C) implantation site (IS) segments from pregnant uteri. Representative sections from (D) non-bead (NB) and (E and F) bead-induced deciduoma (BID) segments of pseudopregnant uteri. AM, anti-mesometrial side; B, bead stimulus; C, conceptus; MM, mesometrial side. Numbers above scale bars are in microns. Sections are representative of at least three independent samples. Global linear adjustments of the brightness and color level were made on the photomicrographs to more accurately represent what was observed on the slides under the microscope.
Citation: REPRODUCTION 141, 4; 10.1530/REP-10-0358
qRT-PCR to assess mRNA levels between the day 7.5 non-implantation (NIS) and implantation (IS) segment tissues of pregnant mice.
| Symbol | NIS | IS | P value |
|---|---|---|---|
| Dact1 | 1.9±0.42 | 1.0±0.15 | >0.05 |
| Hoxb6 | 17.8±4.20 | 1.0±0.13 | <0.005 |
| Nkg7 | 0.3±0.02 | 1.0±0.15 | <0.005 |
| Prss28 | 9.7±2.74 | 1.0±0.37 | <0.001 |
Values represent the mean (±s.e.m.; N=4) relative mRNA levels normalized to 18S rRNA levels.
Discussion
Previous work has compared gene expression in the pregnant uteri to uteri undergoing artificially induced decidualization in an effort to identify genes whose expression levels may be modulated by specific paracrine signals from the conceptus. As a consequence, several genes that are differentially expressed in pregnant mouse uteri during decidualization compared with those undergoing artificially induced decidualization have been identified (recently reviewed in Herington & Bany (2009)). However, these experiments mainly involved the use of either pseudopregnant or ovariectomized hormone-sensitized mice given injections of oil into the uterine lumen to artificially induce decidualization. Recently, we have found that the decidual changes of various artificial models may vary with respect to how they mimic the processes that occur in the pregnant uterus during decidualization (Herington et al. 2009). The BID model appears to be more ‘physiological’ than the more commonly used models that use the injection of sesame oil as the deciduogenic stimulus. For example, the pseudopregnant bead-induced model clearly causes the formation of a functional primary decidual zone similar in timing and location to that of pregnant uteri (Herington et al. 2009). This is not the case for uteri undergoing oil-induced decidualization (Wang et al. 2004, Herington et al. 2009). More importantly, published data suggest that the progression of decidualization in the BID model is more similar to pregnant uteri than that in oil-induced models (Herington et al. 2009). Therefore, in this study, a large-scale assessment of the differences in uterine gene expression between the pregnant uterus and those of pseudopregnant mice undergoing bead-induced decidualization was made. Day 7.5 of pregnancy and pseudopregnancy was the chosen time point since it is well after the process of decidualization begins but is before its completion. Furthermore, prior Affymetrix microarray work comparing gene expression in the day 7.5 decidua compared with oil-induced deciduoma indicated that up to 10% of the genes expressed in the mouse uterus may be impacted by the presence of a conceptus (Kashiwagi et al. 2007). In contrast, using the pseudopregnant BID model, our work found that <0.35% of the genes expressed in the mouse uterus during decidualization may be impacted by the presence of a conceptus. We cannot fully explain this discrepancy between our Illumina platform microarray data and the previously published Affymetrix results. A comparison to the Affymetrix microarray data cannot be done since, to our knowledge, the complete dataset of this work has not been made publicly available. Nonetheless, the bead-induced model appears to be more physiologically relevant than the oil-induced deciduoma model (Herington et al. 2009). It is possible that this is the reason we found significantly fewer differentially expressed genes in this study. However, another possible reason for the lack of identifying a larger number of differentially expressed genes was that our data may have a high false-negative rate. We mainly focus on the set of the genes identified in this study whose expression levels and localization in the uterus may be controlled by signals from the conceptus. However, expression levels of other genes were also studied to verify the lack of differential expression.
Retinoic acid plays a key role in mammalian reproduction and development (reviewed in Clagett-Dame & DeLuca (2002)) but since vertebrates cannot synthesize vitamin A (retinol or retinal) de novo, there is a dependence on a source of key dietary provitamin A carotenoids for the production of retinoic acid and related molecules (reviewed in von Lintig et al. (2005)). Depending on the vitamin A levels and timing of the diets, different negative reproductive outcomes occur in rats at different stages of pregnancy (Clagett-Dame & DeLuca 2002). Although it is assumed that most vitamin A is produced from dietary provitamin A carotenoids within the intestines of mammals (Olson 1961, Fidge et al. 1969, Duszka et al. 1996, During et al. 1998), it has been noted that the major provitamin A form called β-carotene (Heinonen 1991, Granado et al. 1996, Strobel et al. 2007) may be stored in various tissues including the uterus and ovary. β-Carotene found in the uterus may have direct antioxidative effects or it may be converted into vitamin A. As it is mostly responsible for cleaving β-carotene to retinal in mice (Fierce et al. 2008), uterine BCMO1 may be responsible for directing locally stored β-carotene toward retinoic acid at the maternal–conceptus interface. In this study, the expression of the Bcmo1 gene appears to increase solely in the implantation segments of the uterus. Furthermore, we confirmed previous work (Paik et al. 2001) that Bcmo1 is expressed in the maternal tissue near the implanting conceptus undergoing decidualization on days 7.5 and 8.5 of pregnancy. Finally, we found differences in the levels and/or localization of this expression in the BID tissue. This suggests the conceptus may somehow influence Bcmo1 expression in the endometrium during decidualization. Notably, Bcmo1 expression is under the control of peroxisome proliferator activator receptors (PPARs) in other tissues (Boulanger et al. 2003). PPARs play a role in early pregnancy and placentation (reviewed in Lim & Dey (2002) and Huang (2008)), and previous results showed that Ppard expression in the rodent uterus occurs in a region mesometrially to the implanting conceptus (Ding et al. 2003) which is similar to what we show for Bcmo1. More work is required to determine whether PPARD is responsible for driving Bcmo1 expression in the uterus during implantation.
Previous work has revealed that several genes involved in retinoic acid metabolism and action are expressed in rodent uteri and developing placenta during implantation (Sapin et al. 1997, Zheng & Ong 1998, Vermot et al. 2000, Zheng et al. 2000) or decidualization, many of which appear to be controlled by progesterone (Jeong et al. 2005). Retinoic acid might play a role in implantation, placentation, and decidualization since trophoblast and endometrial cells exhibit the expression of retinoic acid-inducible genes during implantation (Sapin et al. 2000). For example, Yan et al. (2001) had shown that retinoic acid promotes the differentiation of trophoblast stem cells to giant cells. Prolactin-like protein expression in the developing placenta may also be influenced by retinoic acid (Lu et al. 1994). Finally, retinoic acid can suppress endometrial stromal cell decidualization (Brar et al. 1996). Bcmo1 expression in the mouse uterus during implantation shown in this study suggests that local retinal production from locally stored β-carotene may play a role in implantation. However, since Bcmo1 knockout mice appear fertile (Lindshield et al. 2008), it is doubtful that the local conversion of β-carotene to retinal by decidual cell BCMO1 is absolutely required for mice to have offspring.
Lipid peroxidation at the fetal–maternal interface is likely counterbalanced by maternal protective measures to have an uncomplicated pregnancy. Lipid peroxidation products increase in patients that have complications such as gestational diabetes and preeclampsia during the late phases of pregnancy (Little & Gladen 1999). Even during normal pregnancy, there is an increase in circulating lipid peroxides, especially in the second trimester (Little & Gladen 1999). Since harmful effects of molecules produced by lipid peroxidation can be serious, it is of considerable interest to understand the mechanisms that limit the effects of lipid peroxides generated at the fetal–maternal interface. In this study, we show that Aldh3a1 expression increases in the maternal tissue surrounding the implanting mouse conceptus in the days leading up to mid-pregnancy. The levels and/or localization of this expression, but not that of the related gene Aldh3a2, in the endometrium during decidualization appeared to be partially dependent on the presence of a conceptus. ALDH3A1 is an aldehyde dehydrogenase that can break down many of the aldehyde products produced by lipid peroxidation (Townsend et al. 2001). Therefore, we speculate the maternal expression of Aldh3a1 observed in this study may be important to prevent the potential harmful effects of lipid peroxidation products that may be generated at the fetal–maternal interface. More work will be required to determine the precise function of Aldh3a1 expression in the uterus during pregnancy.
Inhbb expression in the uterus during implantation may differ between species that are considered to exhibit decidualization. The role of inhibins, activins, and follistatin in the uterus during implantation and decidualization has been extensively reviewed elsewhere (Ethier & Findlay 2001, Jones et al. 2002, 2006). Briefly, INHBB may form homodimers to form activin B or may form heterodimers with INHBA or INHA to form activin AB or inhibin respectively. Inhbb gene expression in the human uterus appears to be associated with the onset of decidualization. Expression is found in stromal cells undergoing decidualization as well as in immune and glandular epithelial cells with the levels of expression being significantly increased in the stroma of the first semester decidua (Jones et al. 2000). Furthermore, the expression of Inhbb, but not the related genes Inhba or Inha, significantly increases in human endometrial stromal cells during decidualization in vitro (Horne et al. 2008). Several lines of evidence suggested that Inhbb is not expressed in the rat endometrium during decidualization (Tessier et al. 2003), but rather Inha expression is transiently increased in the primary decidual zone (Gu et al. 1995). Unlike the rat uterus, this study shows that Inhbb expression increases in the mouse uterus in areas undergoing decidualization. Furthermore, the observation that Inhbb mRNA levels were higher in the BID relative to implantation segments of the uterus leads to the speculation that the presence of a conceptus somehow reduces the increased expression of endometrial Inhbb that accompanies decidualization. Notably, reduced uterine Inhbb expression and decidualization is observed in women with ectopic pregnancies suggesting that expression may be dependent on decidualization in humans (Horne et al. 2008). However, this is unlikely to explain the differences in expression between the IS and BID tissues observed in this study, as previous work has shown that the degree of decidualization is similar between these tissues (Herington et al. 2009). One in situ hybridization study described the lack of Inhbb expression in the mouse uterus during implantation (Manova et al. 1992). However, in a second study, Inhbb expression was localized to decidual cells adjacent to the uterine lumen away from the position of the mouse embryo (Feijen et al. 1994). Therefore, our results localizing Inhbb mRNA to endothelial cells of the vascular islands in the mesometrial decidua and deciduoma are novel. We cannot explain with certainty why discrepancies exist between these three studies. However, expression of Inhbb in vascular endothelial cells has previously been detected (Viemann et al. 2006, Sha et al. 2007, Sobrino et al. 2009). Further work is required to better understand Inhbb expression and function in the mouse uterus during decidualization.
Guca2a and Guca2b genes encode proteins, GUCA2A (guanylin) and GUCA2B (uroguanylin), that bind to a membrane protein called GUCY2C (Sindic & Schlatter 2006a). GUCY2C is a membrane guanylate cyclase, and upon the binding of GUCA2A or GUCA2B there is an increase in cGMP levels within the cells. Recent work has shown that the expression of Guca2b in the rat uterus increases during pregnancy then dramatically decreases just prior to parturition (Girotti & Zingg 2003). In this study, we found that Guca2b expression is dramatically increased throughout the mouse uterus during decidualization and is differentially expressed between the IS and BID segment tissues. The reason for this differential expression as well as the function of the Guca2b expression in the uterus during rodent pregnancy is not completely understood. It has been speculated that GUCA2B may act on the myometrium and cause myometrial quiescence during pregnancy by increasing cGMP levels (Girotti & Zingg 2003). However, to the best of our knowledge, the expression of Gucy2c in the rodent myometrium has not been described in the literature, and we were not successful in detecting expression in the mouse uterus during decidualization. Regardless, a Guca2b DNA vaccine has been shown to have anti-fertility effects in mice (Sun et al. 2008). It remains to be determined whether the anti-fertility effect is specifically due to altered uterine GUCA2B action. Finally, if Gucy2c is not expressed in the uterus, it seems plausible that GUCA2B may function only as an endocrine factor produced in the uterus during pregnancy. Alternatively, if Gucy2c is expressed in the conceptus, uterine GUCA2B might be a paracrine factor and exert effects on the conceptus. Future studies interrogating the function of Guca2b expression in the uterus during the peri-implantation period are warranted.
Paracrine signals from trophoblast cells are believed to control the expression of endometrial gene expression in several ungulate species and humans, but evidence for this is less obvious for rodents (reviewed in Herington & Bany (2009)). Since trophoblast giant cells are the part of the conceptus in most intimate contact to the maternal tissue, we have previously hypothesized that these cells may secrete paracrine factors that regulate uterine gene expression during decidualization (Bany & Cross 2006). However, it seems that signals from the trophoblast giant cells are not required for the differential expression of Aldh3a1, Bcmo1, Inhbb, and Guca2b observed in this study, as expression levels are not different between implantation segments containing Hand1 knockout compared with wild-type conceptuses. In fact, we have not been able to identify differential expression between such samples by microarray analysis (BM Bany, unpublished data). This suggests that up to day 7.5 of pregnancy, the giant cells may have very little effect on endometrial gene expression during decidualization in the mouse. We speculate that paracrine factors from other components of the conceptus may be responsible for the differential expression observed in this study. Alternatively, the difference between the physical stimuli of a growing conceptus to that of a fixed blastocyst-sized bead may be responsible. Furthermore, whether giant cells exert paracrine effects on the endometrium during later days of pregnancy as the major phases of placental development occur still needs to be determined.
In conclusion, we have demonstrated differential gene expression in the uterus during decidualization in pregnant mice compared with bead-induced pseudopregnant mice. This differential gene expression supports a previous suggestion that signals from the rodent conceptus may influence uterine gene expression during decidualization (Herington et al. 2009). However, the much lower magnitude of the differential expression observed in this study is in contrast to previous microarray work (Kashiwagi et al. 2007), where the oil-induced deciduoma model was used. These differences may be explained, at least in part, to recent observations that the BID model more closely mimics pregnancy than the other commonly used deciduoma models (Herington et al. 2009). Notably, this study provides the identity of several genes whose expression in the uterus during decidualization may be influenced by molecular or physical signals from the conceptus.
Materials and Methods
Animals and tissue collection
All procedures involving mice were approved by Southern Illinois University Institutional Animal Care and Use Committee. Female (9–12 weeks old) and male (3–10 months old) CD1 mice (Charles Rivers Laboratory, Wilmington, MA, USA) were housed under controlled light conditions (lights on: 0700 to 1900 h) and allowed free access to food (Formulab 5008 LabDiet; Purina Mills, Gray Summit, MO, USA) and water. Females were mated with fertile males, and the morning a vaginal plug was observed was termed day 0.5 of pregnancy. The model used to obtain artificially induced deciduomas for this study has been described in detail elsewhere (Herington et al. 2009). Briefly, female CD1 mice were mated with vasectomized males, and the morning a vaginal plug was observed was considered day 0.5 of pseudopregnancy. On day 2.5, between 1300 and 1500 h, blastocyst-sized concanavalin A-coated agarose beads (Sigma) were transferred into the uterine lumen. This BID model was chosen over other artificial decidualization models, because it better recapitulates the decidual changes that occur in the pregnant uterus compared with other commonly used models (Herington et al. 2009).
Animals were killed at 0900 h on days 3.5–8.5 of pregnancy or pseudopregnancy for tissue collection. The uterine horns were dissected and cleared of mesentery. In the case of uteri collected on day 4.5 of pregnancy or pseudopregnancy, implantation and bead-containing segments of the uterus were visualized by an i.v. injection of Evans blue dye as described previously (Herington et al. 2009). For days 4.5–8.5 uterine horns, the IS or BID segments were separated from NIS or NB segments respectively. For implantation segments, all tissues of the conceptus (embryo and trophoblast cells) were carefully dissected out and discarded. All the remaining tissue were then pooled and considered IS segment tissue.
To assess the potential function of giant cells on conceptus-dependent gene expression levels in the uterus, Hand1 mutant mice were used. Hand1-deficient conceptuses fail to develop past days 7.5–9 of pregnancy, and the failure of the conceptus appears to be due to lack of trophoblast giant cells (Riley et al. 1998). Heterozygous animals were intercrossed to obtain timed pregnant female mice carrying wild-type, heterozygous, and knockout conceptuses and used as described previously (Bany & Cross 2006). PCR genotyping of the embryos was carried out by PCR as described by Riley et al. (1998).
RNA extraction
Tissues were homogenized in TRIzol reagent, and total RNA was extracted as recommended by the manufacturer (Invitrogen). The total RNA was treated with Turbo DNAse as recommended by the manufacturer (Applied Biosystems, Foster City, CA, USA) and was then re-extracted with TRIzol reagent. Total RNA concentrations were measured spectrophotometrically, and samples were subjected to denaturing agarose gel electrophoresis to verify RNA integrity and purity.
Illumina microarray analysis
This study was to extend the findings of previous work (Bany & Cross 2006, Kashiwagi et al. 2007) that directly compared the development and function of decidua and deciduoma at 3 days after the onset of decidualization. Therefore, RNA isolated from the uterine tissue from day 7.5 IS and BID segments were used for the microarray analysis (N=3). The RNA quality was further verified using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) before microarray analysis. The total RNA was then used for microarray analyses using the Illumina platform and a whole-genome Expression BeadChip array slide (MouseWG-6 v1.1; Illumina, Inc., San Diego, CA, USA) to determine differences in steady-state mRNA levels between the tissues. The technology used in this platform involves microbeads coated with oligonucleotide probes and is described in detail elsewhere (Fan et al. 2006). Briefly, each MouseWG-6 v1.1 slide contains six separate whole-genome gene expression arrays, allowing six target samples to be hybridized to six independent arrays on one slide. Each array contains over 46 000 different oligonucleotide probes. Target preparation, hybridization, and scanning were conducted by the Microarray Facility at the Hamilton Eye Center (UT Memphis) using the exact methods suggested by the manufacturer (Illumina, Inc.). Briefly, total RNA isolated from the tissue for each experiment was used to create six biotin-labeled cRNA target samples (N=3) using the Illumina RNA amplification kit (Applied Biosystems). The cRNA target preparations were then hybridized to the arrays for 16–20 h at 55 °C, and the bound biotin-labeled targets were then detected using streptavidin-Cy3 and an Illumina BeadArray Reader with BeadStudio software (Illumina, Inc.).
Data analysis
Complete microarray data can be found at NCBI's GEO database (GSE25903, www.ncbi.nlm.nih.gov/geo). Raw intensity data were background subtracted and filtered to include only those with detection scores >0.98 (P<0.02) for all samples. The data were then exported to Chipster for further analysis (Chipster.csc.fi). Chipster, developed by the Finnish IT Center for Science CSC (Chipster.csc.fi), is a useful graphical user interface to a collection of online microarray analysis tools such as R/Bioconductor (Gentleman et al. 2004). The scale normalization (Smyth & Speed 2003) tool of Chipster, which uses the limma R package, was used to normalize the data. Significance of differential expression was determined using two different methods. First, the Chipster tool that uses the Bioconductor package limma Empirical Bayes method (Smyth 2004) using the default Benjamini–Hochberg method to control the false discovery rate was used. Differences were considered significant if the adjusted P values were <0.05. In the second analysis, the Reproducibility-Optimized Test Statistic (ROTS) tool was used with a cutoff false discovery rate of <0.05. The ROTS procedure enables the selection of a suitable gene ranking statistic directly from the given dataset and is described in detail elsewhere (Elo et al. 2008, 2009). The lists of differentially expressed genes were combined and annotated using Bioconductor annaffy package. The hypogeometric test for GO tool of Chipster was used to determine over-and underrepresented go terms of the list of differentially expressed genes using a cutoff of P<0.01 for statistical significance. Finally, a heat map was constructed using the clustering tool of Chipster to visually show signal levels detected for each sample of the differentially expressed genes.
Quantitative real-time PCR
qRT-PCR was carried out as described previously (Herington et al. 2009) using oligonucleotide primers (IDT Technologies, Coralville, IA, USA) shown in Supplementary Table 3A. Briefly, High Capacity RNA-to-cDNA kits (Applied Biosystems) were used to generate the cDNA. PCR was performed using 2x iQ SYBR Green Supermix and a CFX real-time PCR machine (Bio-Rad). PCR conditions were 3 min at 94 °C followed by 40 cycles of 94 °C, 62–65 °C for 20 s, and 72 °C for 1 min for melting, annealing, and extension steps respectively. The PCR primers were used at a final concentration of 200 nM. Cycle thresholds (Ct) were determined using the CFX software, and relative mRNA levels were determined as described previously after normalization to 18S rRNA levels (Herington & Bany 2007a). The efficiencies of the PCR were linear between the mRNAs and 18S rRNA and were >85%. Differences between mean levels of the steady-state mRNA levels were determined statistically as described previously (Herington & Bany 2007a).
In situ hybridization
To collect tissue for in situ hybridization, mice were anesthetized with an i.p. injection of a ketamine hydrochloride (200 mg/kg) and xylazine (20 mg/kg; Henry Schein, Melville, NY, USA). Perfusions of PBS and fixative were then performed as described previously (Herington & Bany 2007b). Uteri were then dissected out, and uterine samples were immersed in fresh 4% (w/v) paraformaldehyde in PBS for 24 h, then in 70% (v/v) ethanol for 24 h at 4 °C. Processing of tissue into paraffin was done using routine methods and then 5 μm sections were mounted onto HistoBond glass slides (Statlab, McKinney, TX, USA).
To prepare templates for sense and antisense riboprobe synthesis, PCR amplicons were generated using TopTaq DNA polymerase and purified using QIAquick PCR purification kits as recommended by the manufacturer (Qiagen) using the primers shown in Supplementary Table 3B. The amplicons were then cloned into the pGEM-T Easy vector (Promega), which contains SP6 and T7 RNA polymerase sites flanking the multiple cloning region. After sequencing the cDNA clones, the plasmids were used to generate transcription templates by PCR using TopTaq (Qiagen) and M13 forward or reverse primers plus the appropriate insert-specific primer. After purification of the DNA using a QIAquick PCR purification kit (Qiagen), the template was used to generate digoxigenin (DIG)-labeled riboprobes using MAXIscript T7 and MAXIscript SP6 kits (Applied Biosystems) along with dixoxigenin-11-UTP (Roche). After riboprobe purification using MEGAclear kits (Applied Biosystems), in situ hybridization was performed using antisense DIG-labeled riboprobes exactly as described in detail elsewhere (Simmons et al. 2007). Both antisense and control sense DIG-labeled riboprobes were used for in situ hybridization. Use of sense probes for all target genes studied in the in situ hybridization work of this study did not result in any positive hybridization signals (data not shown).
Supplementary data
This is linked to the online version of the paper at http://dx.doi.org/10.1530/REP-10-0358.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported in part by the Southern Illinois University School of Medicine and an NIH Grant (HD049010) from the Eunice Kennedy Shriver National Institute of Child Health and Human Development. The microarray hybridization and scanning core facility used for the work in this study was supported by an NIH National Eye Institute Vision Core Grant (PHS P30 EY013080).
Acknowledgements
We would like to thank Ms Sheila Scillufo for her technical help on this project.
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