Division of Genetics (M.W.M.Y., S.E.C., A.F., Y.D., S.M., R.L.M.), Department of Medicine and Department of Obstetrics and Gynecology (M.W.M.Y., D.J.S.), Brigham and Womens Hospital, Harvard Medical School, Boston, Massachusetts 02115; Departments of Obstetrics and Gynecology, and Cell Biology and Physiology (H.L.), Washington University School of Medicine, St. Louis, Missouri 63110; and Department of Genetics (S.E.C., G.M.C.), Harvard Medical School, Boston, Massachusetts 02115
Address all correspondence and requests for reprints to: Richard Maas, M.D., Ph.D., Division of Genetics, Department of Medicine, Brigham & Womens Hospital and Harvard Medical School, Thorn Building, Room 1019, 20 Shattuck Street, Boston, Massachusetts 02115. E-mail: maas{at}rascal.med.harvard.edu.
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ABSTRACT |
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INTRODUCTION |
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At the same time, gene targeting studies show that implantation of a healthy embryo can fail due to a defective uterine environment. Priming of the uterine stroma by progesterone (P4) is essential for the establishment of an appropriate uterine environment for implantation. Although knowledge of the molecular pathways that act downstream of P4 in implantation is limited, one gene that is both P4 responsive and required for the establishment of an appropriate environment for embryo implantation in the mouse uterus is Hoxa-10, a member of the AbdB subclass of Hox genes (4, 5, 6). Although Hox genes are well known as regulators of patterning in both vertebrate and invertebrate embryonic development, Hoxa-10-deficient adult female mice exhibit a severe failure of implantation and defective decidualization that lead to recurrent pregnancy loss and infertility (6).
The uterus is comprised of three major cellular compartments: epithelium, stroma, and myometrium, which are under differential hormonal regulation by ovarian estrogens and P4 (2, 3). In murine reproductive physiology, the major preovulatory ovarian estrogen, estradiol (E2), stimulates the uterine epithelium on d 0.5 to 1.5 post coitum (p.c.), whereas P4 stimulation of the uterine stroma is first evident by d 2.5 p.c. On d 3.5 p.c., estrogen levels rise again so that the uterine stroma has been sequentially primed by estrogen and P4 and is now regulated by both hormones simultaneously (2).
Hoxa-10 is up-regulated by P4 in the uterine stroma at d 3.5 p.c., 24 h before implantation, and its expression persists in the developing decidua (5, 7). Moreover, previous studies have shown that Hoxa-10 is required for P4 responsiveness in the uterine stroma (8). For example, in a model in which Hoxa-10 mutant females are ovariectomized to eliminate variability in hormonal cycling between animals, and then treated with estrogen and P4, stromal cell proliferation is impaired, whereas epithelial cell proliferation is unaffected. In this assay, exogenous P4 and estrogen greatly amplify the magnitude of the basal uterine stromal proliferation defect in ovariectomized Hoxa-10 mutants relative to wild type (8).
Hox genes have been proposed to act as local regulators of cell proliferation during development (9, 10), but molecular effectors for their proposed cell cycle-regulatory function have not been identified. Based on the observed proliferation defect in the Hoxa-10 mutant stroma, we reasoned that P4 administration to ovariectomized wild-type and Hoxa-10-deficient females should amplify differences in gene expression that underlie the Hoxa-10 mutant stromal proliferation defect. We therefore employed global gene expression profiling to identify Hoxa-10 downstream genes that mediate the stimulatory effect of P4 on cell cycle progression in the uterine stroma. At the same time, we sought to gain insight into other P4-dependent downstream events that contribute to the defective implantation phenotype in Hoxa-10 mutant females.
Although implantation depends upon the precisely coordinated effects of estrogen and P4, to study the effects of one steroid hormone at a time, we initially chose to focus only on the gene expression changes produced by P4. However, to establish physiological relevance of findings derived from an ovariectomy model to normal implantation, we then further evaluated the results in a combined E2 + P4 context, d 3.5 p.c. of natural pregnancy.
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RESULTS |
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Gene Expression Profiling Detects Differentially Regulated Genes (DRGs) in the Hoxa-10 Mutant Uterine Stroma
Gene expression profiles of more than 12,000 genes (6,000 known genes and 6,000 expressed sequence tags) represented on the U74Av.2 oligonucleotide array (Affymetrix, Santa Clara, CA) were first analyzed by dChip (11, 12). All subsequent analyses were performed on dChip model-based expression indices. The fold difference cutoff in all analyses was 1.5-fold unless otherwise specified. Independent analyses using Statistical Analysis of Microarrays (SAM) and conventional t test (13, 14) yielded 92 and 81 genes, respectively, that were differentially expressed between wild-type and Hoxa-10 mutant uteri in the OVX/6 h/P4 model (Table 1). A total of 57 genes were identified by both SAM and t test. The high degree of overlap (>50%) between these two analytical methods, which are based on very different statistical assumptions, points to the robustness of the data analysis. Combining the gene lists obtained by SAM and t test yielded a total of 116 differentially regulated genes, or DRGs, and all subsequent data analyses and experiments were based on these 116 DRGs (Table 1
).
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Four such clusters are shown in Fig. 3. Cluster 3 demonstrates the consistency of the data and the power of this analytical method in clustering coregulated genes. Of the genes in cluster 3, 78% (40/51) consisted of Ig genes, which are B lymphocyte specific (Fig. 3A
). Interestingly, there was no apparent difference in the regulation of these Ig genes between wild type and mutant. Nonetheless, the striking coordinate down-regulation of Ig genes in response to P4 indicates that major changes in the dynamics of intrauterine B cell trafficking, Ig gene expression, or both, occur in response to P4.
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Uterine Stromal Cell (USC) Proliferation Is Inhibited in Hoxa-10 Mutants
To test whether stromal cell proliferation was disrupted on d 3.5 p.c. of natural pregnancy in the Hoxa-10 mutant uterus, we injected d 3.5 p.c. pregnant wild-type and mutant mice with BrdU, and 4 h later, performed immunostaining on freshly isolated USCs with anti-BrdU antibody. The immunostained cells were then analyzed by flow cytometry. In wild-type mice, 8.9% ± 0.1% (mean ± SEM) of USCs were BrdU+, with 5% background fluorescence in unstained control cells. However, in four replicate experiments in the Hoxa-10 mutant, only 5.7 ± 0.2% of USCs were BrdU+ (Fig. 4C). The decrease in the proportion of stromal cells that incorporated BrdU in the Hoxa-10 mutant was significantly lower than in wild type (P < 0.01). The decrease in S-phase entry by mutant USCs indicates an important cell cycle-regulatory function of Hoxa-10 that is compatible with the altered expression of p57 and p15 in the Hoxa-10 mutant periimplantation uterus.
Expression Profiling Detects Abnormal Accumulation of T Lymphocytes in the Hoxa-10 Mutant Uterus
We next investigated the biological relevance of a second group of DRGs that was expressed at higher levels in the Hoxa-10 mutant uterus than in wild type: those pertinent to immune function. In particular, genes expressed in T lymphocytes, including TCRV4, TCR
C, and MHC class II I-Ab, were overexpressed in the Hoxa-10 mutant uterine expression profile. Although this result could reflect increased expression of these genes on a per cell basis, we first chose to test the hypothesis that T lymphocytes, a distinct cell population in the uterine stroma, were increased in number in the Hoxa-10 mutant. Indeed, flow cytometry of cells isolated from the uterine stroma of d 2.5 and 3.5 p.c. pregnant mice, with splenocytes as a control, indicated that the proportions of CD4+ and CD8+ T cells were significantly increased in the mutant uterine stroma (Fig. 5A
). In addition, this expansion of T cells was polyclonal, as evident by increases in TCR
, TCR
ß, and NK lineages in the Hoxa-10 mutant uterus on d 3.5 p.c. (Fig. 5
, A and B). Consistent with microarray results showing increased expression of TCR
V4 and TCR
C in the Hoxa-10 mutant, the increase in the number of
T cells in the physiological pregnant state occurred earliest and was the most dramatic (Fig. 5A
). Microarray analysis of heterogeneous uterine tissue thus revealed alterations in gene expression that at least partly reflected changes in cell number within specific stromal T cell subpopulations. These analyses thus indicate a significant and previously unsuspected immunological phenotype in the Hoxa-10 mutant uterus.
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We performed immunostaining experiments to further confirm the localization of T cells in the uterus. We chose the CD4 marker for these localization experiments as CD4+ cells were twice as abundant as CD8+ cells in all flow cytometry experiments, as seen in the representative experiment in Fig. 5B. Immunostaining experiments confirmed that the CD4+ cell population resided mainly in the stromal compartment in both wild-type and Hoxa-10 mutant uterus at both d 0.5 p.c. and d 3.5 p.c. (Fig. 5C
). On d 0.5 p.c., when estrogen effects predominate, there was no overt difference in CD4+ abundance between wild-type and Hoxa-10 mutant uterus. At d 3.5 p.c., however, when P4 levels are high, CD4+ immunostaining was greater in the mutant stroma than in wild type (Fig. 5C
). The d 3.5 CD4+ immunostaining results are thus consistent with the CD4+ flow cytometry analyses (Fig. 5A
).
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DISCUSSION |
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An attenuation of P4-induced Hoxa-11 expression was identified in the Hoxa-10 mutant uterus by both microarray and real-time RT-PCR. This result was previously not identified by in situ hybridization (8), probably because of the inherently nonquantitative nature of this technique. Cross-regulation of Hox genes in embryonic patterning is well known (21), as exemplified by the regulation of Hoxb2 by Hoxb1 in rhombomere r4 during mouse embryonic development (22). The cross-regulation of Hoxa-11 by Hoxa-10 also has important implications in implantation, including the possibility that the progesterone receptor (PR) and Hoxa-10 functionally cooperate to mediate the full induction of Hoxa-11 stromal expression. The relevance of Hoxa-10 and Hoxa-11 regulatory interactions to the study of human infertility is further supported by the finding of up-regulated HOXA-10 and HOXA-11 expression in the human uterus in response to P4 during the time of implantation (23).
Comparison to Other Microarray Studies
The experimental approach described here differs from that in other microarray studies designed to identify genes involved in implantation (24, 25, 26). In one study, the same Affymetrix microarray (U74A version 2) used in our analyses was used to identify genes that are differentially expressed between implantation and nonimplantation sites in the wild-type uterus at 2324 h of d 3 p.c. (26). In our study, we employed unopposed P4 stimulation in wild-type and Hoxa-10 mutant females to identify Hoxa-10-dependent DRGs in the uterine stroma. Under these conditions, Hoxa-10 expression is strongly induced throughout the uterine stroma (7), a situation that should favor the identification of P4-inducible Hoxa-10-dependent stromal target genes. Importantly, however, we also tested the in vivo relevance of these DRGs in a physiological pregnancy model at a time point, the morning of d 3 p.c., before implantation and nonimplantation sites are distinguishable. This strategy thus permitted validation of the findings from the OVX/P4/6 h model and should have enriched for Hoxa-10-regulated events that precede overt morphological signs of implantation failure in the mutant.
Thus, although our OVX/P4/6 h model cannot be directly compared with the above-mentioned study, data from our OVX/P4/18 h experiments (data not shown), in which uterine RNA was extracted at 18 h after P4 injection, do detect some of the same DRGs reported by Reese et al. (26). For example, BiP, encoding a calcium-binding Hsp70 class chaperone (reviewed in Ref. 27), is expressed at higher levels in the wild-type than in the Hoxa-10 mutant uterus. The same gene was also found by Reese et al. (26) to be more highly expressed in wild-type implantation sites than interimplantation sites. The expression of genes such as BiP may thus mark a later time point in the P4 response than that addressed by the OVX/P4/6 h model.
An especially interesting point of similarity between the present data and that of Reese et al. (26) relates to the striking, coordinate regulation of some 40 Ig genes in cluster 3 of our SOM analysis. These transcripts are markedly reduced by 3 h after P4 injection, followed by a small peak of expression at 6 h and a slow return to baseline by 24 h. In a composite list of genes showing decreased expression at implantation sites and after initiation of E2-induced implantation, Reese et al. identified 21 immune-related genes, of which 14 are classical Ig genes (Table III in Ref. 26). Strikingly, 10 of those 14 Ig genes are also represented in cluster 3 of our P4 time series experiments, with another three present in the next most closely related cluster, cluster 6. Analysis of sequence similarity among the probe sets representing these genes indicates that their coregulation is unlikely to be explained by cross-hybridization alone (12).
The fact that so many of the same Ig genes were identified in both studies, albeit in different contexts, has interesting implications concerning the role of B cells in implantation. As proposed by Medawar (28), a key requirement for establishment of an appropriate uterine implantation environment, in which the implanting blastocyst resembles an allograft, is the induction of a state of immune tolerance. Although much attention has focused on the role of T cells and cellular immunity during implantation (reviewed in Ref. 29), less is known about the regulation of B cell function. Interestingly, although the PR is expressed in B lymphocytes (30), a prior study did not identify major changes in the number of intrauterine B cells after E2 or E2 + P4 in either wild-type or PR knockout (PRKO) mice (31). Further experiments are required to determine whether P4 mediates local uterine B cell function by regulating Ig family gene expression rather than by controlling B cell number.
Hoxa-10 Is Required for P4-Mediated Stromal Cell Proliferation and CKI Repression
The proliferation of stromal cells and their subsequent differentiation into decidual cells are critical events in periimplantation uterine development. In this regard, a key finding was that two CKI genes, p15 and p57, were aberrantly expressed in the Hoxa-10 mutant uterine stroma. Interestingly, p57 exhibits similar diffuse stromal expression in both wild type and mutant, but its expression is quantitatively more abundant in the Hoxa-10 mutant. In contrast, p15 undergoes a marked shift in its expression from a predominantly myometrial and submyometrial distribution in the wild-type uterus to a diffuse stromal pattern in the Hoxa-10 mutant.
The altered expression of p15 and p57 in the Hoxa-10 mutant is notable. High expression levels of these CKIs during early G0/G1 can induce cell cycle arrest, as p15 and its family members act as specific inhibitors of the cyclin D-dependent kinases cdk4 and cdk6 (32). p57 family members show similar interactions albeit with a broader range of cyclin-cdk complexes (32). The potential functional roles of p15 and p57 are especially relevant as cyclin D3 associates with cdks 4 and 6, and cyclin D3 is the major G1S cell cycle regulator in the periimplantation uterine stroma (33). Interestingly, in the context of myelomonocytic cell differentiation, Hoxa-10 directly up-regulates expression of the CKI p21 and induces differentiation (34). In contrast, in implantation we suggest that it is the quantitative or spatially restricted repression of CKIs such as p57 and p15 by Hoxa-10, be it direct or indirect, that could explain the stromal cell proliferation defect in Hoxa-10 mutants. p57 null mutants die within 10 d of birth and are thus uninformative for the consequences of p57 loss of function in the uterus; p15 knockout mice exhibit no uterine phenotype and are even fertile. However, as these results pertain only to loss of function, the functional significance of the increased CKI expression observed in the Hoxa-10 mutant uterus remains open.
The Hoxa-10 Mutant Uterine Stroma Exhibits Aberrant Lymphoproliferation
A third key finding in the expression profiling experiments was that of increased transcript levels of various T cell genes. Although the absolute expression of these genes on a per cell basis may be altered, the increased number of T cells in the mutant stroma, as demonstrated by flow cytometry, is in qualitative agreement with the increased levels of T cell-related transcripts in the mutant. Moreover, flow cytometry analyses indicated an increased proliferation rate of these cells.
The lymphoproliferation observed in the Hoxa-10 mutant uterine stroma is polyclonal and occurred after both syngeneic and allogeneic matings. These observations argue for a defect in T cell signaling and against an antigen-specific immune response as the cause for the immune phenotype. Hoxa-10 is expressed in early myeloid progenitors but is not known to be present in mature neutrophils, monocytes, or lymphocytes (35, 36). This is consistent with our findings that splenocytes do not express Hoxa-10 (Fig. 1B). Thus, although it remains to be formally tested whether T cells in the periimplantation uterus express Hoxa-10, the available evidence suggests that the aberrant, intrauterine lymphoproliferation in Hoxa-10 mutants is unlikely to result from a T cell autonomous defect. Interestingly, as exemplified by cluster 6 of the P4 time series, many chemokines, chemokine receptors, and cytokines known to be mitogenic for T cells are down-regulated in the uterine stroma in the presence of P4, and many of these same immunoregulatory genes are incompletely repressed by P4 in the Hoxa-10 mutant stroma (12). Thus, it is attractive to propose that in the mutant uterus, Hoxa-10-deficient stromal cells stimulate the inappropriate proliferation of T cells by paracrine signaling mechanisms.
The distinct immunological phenotype in the Hoxa-10 mutant uterine stroma may directly cause the implantation defect in Hoxa-10 mutant females, as the cytolytic and inflammatory activities of T cells are well known to adversely affect the viability of implanting blastocysts. Indeed, the importance of local uterine immunosuppression to normal embryo implantation and pregnancy has been observed in several mouse models. For example, in a CBA/J x DBA/2J mouse model that exhibits a high rate of spontaneous abortion mediated by a monoclonal infiltration of T cells, treatment with a monoclonal antibody directed against the
T cell clone restores fertility (29, 37). Implantation defects are also observed in mouse models with altered uterine immunosuppression (38) and aberrant T cell proliferation (39), and these defects are abolished in lymphocyte-deficient mice, which have normal fertility. Lastly, embryonic resorption and maternal leukocyte infiltration are observed at implantation sites in the Hoxa-10 mutant (6). Thus, the aberrant lymphoproliferation observed in the Hoxa-10-deficient pregnant uterus can be viewed as a localized, proinflammatory response that could potentially compromise pregnancy.
It is interesting to compare the abnormal immune state present in the Hoxa-10-deficient uterus with the proinflammatory uterus described in PRKO mice (40). Experiments using the PRKO mice demonstrate that P4 acts via its receptor to antagonize the proinflammatory activity of estrogen, thereby decreasing the number of neutrophils and macrophages in the uterus. In contrast, the number of B lymphocytes remains unchanged (31). In the Hoxa-10 mutant, our data suggest that P4 may act at the time of implantation through Hoxa-10 to reduce the number of T cells in the uterus. Alternatively, Hoxa-10 may act with the PR to coregulate T cell number; moreover, these possibilities are not exclusive.
Although many possible mechanisms may underlie the preponderance of up-regulated DRGs observed in the Hoxa-10 mutant uterus, repressive functions have been described for both PR (41, 42, 43) and Hox proteins (44, 45, 46). Thus, it is possible that Hoxa-10 may act as corepressor and be required for transcriptional regulation by PR. Repressive functions of Hox genes have been described in Drosophila development (44), and corepressor activity has been described for several mammalian Hox genes (45, 46), but mammalian targets of Hox gene repression, direct or indirect, have been difficult to ascertain in vivo. Based on their differential regulation in the Hoxa-10 mutant uterus, we have identified several candidate Hoxa-10 downstream genes, at least one of which, Hoxa-11, is also required for implantation (47). In addition, the analysis of specific DRGs in the d 3.5 p.c. pregnant uterus suggests a potential mechanism for how Hoxa-10 regulates stromal cell proliferation. Lastly, our analyses at d 3.5 also reveal that Hoxa-10 is required for the proper regulation of intrauterine T cell dynamics. Both of these latter events are critical to implantation.
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MATERIALS AND METHODS |
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Experimental Animals
All animal experimentation described was conducted in accord with accepted standards of humane animal care. Protocols for animal work were approved by the Harvard University Institutional Committee on Animal Care.
Western Blot Analysis
Uteri and spleens were pooled from groups of at least three wild-type or mutant ovariectomized mice at 0, 1, 3, and 6 h after P4 injection and placed in cold RIPA (radioimmunoprecipitation) buffer containing protease inhibitor cocktail (50 µl of 25x protease inhibitor cocktail per ml RIPA buffer, Roche, Indianapolis, IN) on ice. Tissues were then homogenized by polytron, centrifuged at 14 x g at 4 C for 10 min; supernatant protein was quantitated by Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA), and then stored at -80 C. Unless otherwise noted, 25 µg protein samples were analyzed by 10% SDS-PAGE. After nitrocellulose transfer and blocking overnight at 4 C in 5% nonfat milk, membranes were incubated in affinity-purified rabbit polyclonal anti-Hoxa-10 antibody at 1:300 dilution for 1 h at room temperature (RT), washed with 1x Tris-buffered saline with Tween 20 (3 x 10 min), incubated with goat antirabbit (horseradish peroxidase) secondary antibody (Pharmacia Biotech, Piscataway, NJ), washed with Tris-buffered saline with Tween 20, and incubated in enhanced chemiluminescence for 1 min (Bio-Rad Laboratories, Inc.) followed by film development. Hoxa-10 polyclonal antibody was raised in rabbits against the MAP peptide H-EEAHASSSAAEELSPAPSE-8-MAP (Research Genetics, Inc., Huntsville, AL), which corresponded to a sequence in the mouse Hoxa-10 C terminus, and affinity purified using antigen-bound Affygel-10 (Bio-Rad Laboratories, Inc.).
RNA Isolation and Oligonucleotide Microarray Hybridization
Groups of four wild-type and four Hoxa-10 mutant mice (SvJ background) at 1016 wk of age were ovariectomized. Fourteen days later, P4 (Sigma, St. Louis, MO) dissolved in sesame oil (Sigma) was injected sc (2 mg/mouse in 100 µl), after which the animals were killed and uterine horns removed at 6 h after injection. Care was taken to exclude the region of homeotic transformation at the uterine-oviductal junction (proximal 25% of each uterine horn) (3). Fat and mesentery were trimmed. Tissues were snap frozen in liquid N2, pooled, and homogenized by mortar in N2. Total RNA was extracted using Trizol (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturers protocol, quantified by UV spectroscopy, and stored in diethylpyrocarbonate water (Ambion, Inc., Austin, TX) in 1-µg/µl aliquots at -80 C. A portion (0.5 µg) of each RNA sample was analyzed on a 2% agarose gel to confirm integrity. Prechip validation of RNA collected via the OVX/P4/6 h protocol was performed by assaying for up-regulation of Hoxa-10, Hoxa-11 (7), and Histidine decarboxylase (48) as markers of P4 efficacy.
Reverse transcription used oligo-dT followed by in vitro transcription and biotin labeling of cRNA (Enzo Biochem, Farmingdale, NY). All cRNA samples were analyzed with a Bioanalyser 2100 (Agilent Technologies, Wilmington, DE) before chip hybridization. Fragmented, labeled cRNA (20 µg) was hybridized to Affymetrix U74A version 2 mouse oligonucleotide arrays, which were washed and scanned per Affymetrix protocol. Control samples were prepared and analyzed similarly except that sesame oil vehicle (OVX/oil/6 h) rather than P4 was injected. Triplicate pairs of wild-type and mutant samples were prepared, and in vitro transcription was performed in parallel for each of OVX/P4/6 h and OVX/oil/6 h RNA collections.
Data Analysis for OVX/P4/6 h Wild-Type vs. Mutant Comparison
Intensity and cell data were obtained using MAS 4.0.1 (Affymetrix). Data were normalized and analyzed using dChip (11) to obtain model-based expression levels for each probe set, upon which all subsequent analyses were performed. Criteria for significant differential expression levels between wild type and mutant were: a fold change in expression of at least 1.5, P value of at least 0.1 (based on a t test or other tests), and an absolute difference in expression of at least 75 U. Paired and unpaired t tests were performed using Excel (Office 2000, Microsoft Corp.). Expression levels from dChip were also analyzed by SAM, an Excel add-in developed by Tusher et al. (13), that uses a variant of the t statistic and a permutation analysis to estimate the false positive rate within a set of significantly up- or down-regulated genes. These false positive rates take multiple hypothesis testing into account. When using paired tests with a 1.5-fold cutoff threshold and a median false detection rate of 10%, the SAM algorithm yielded similar lists of genes to those using conventional t statistics. DRGs were classified according to their described functions based on a literature search of PubMed (http://www.ncbi.nlm.nih.gov/PubMed/). A DRG was placed in more than one category if multiple functions are described.
Real-Time RT-PCR
Separate aliquots of total RNA from the chip experiments were stored at -80 C. In the physiological pregnancy model, d 3.5 p.c. total uterine RNA was pooled from one or two wild-type or mutant mice. Samples were treated with deoxyribonuclease I using the DNA-free kit (Ambion, Inc.) and diluted to 20 ng/µl. Reverse transcription and quantitative PCR were performed with Superscript II reverse transcriptase (RT) and Taq polymerase in the One Step RT-PCR kit (Life Technologies, Inc.) on the iCycler (Bio-Rad Laboratories, Inc.). No RNA and negative RT controls (no RT, Taq polymerase only) were included in each set of RT-PCR experiments. All samples that would be compared were tested in the same RT-PCR run. Primer and fluorescence resonance energy transfer probe sequences were designed using Primer Express 1.0 (PE Applied Biosystems, Norwalk, CT), purchased, or extracted from published literature. All primers were from Invitrogen (San Diego, CA) and all fluorescence resonance energy transfer probes with 5'-FAM and 3' black hole quencher labels were from BioSource Technologies, Inc. (Vacaville, CA). Sequences of primer and probe sets are listed in the supplemental data (Ref. 12 and published on The Endocrine Societys Journals Online web site, http://mend.endojournals.org). Threshold cycle numbers were obtained using iCycler software version 2.3 (Bio-Rad Laboratories, Inc.). Conditions for amplification were: RT cycle at 50 C for 15 min, 1 cycle of 95 C for 2 min, 35 cycles of 95 C for 15 sec, 59 C for 15 sec, and 72 C for 15 sec. Quantitative RT-PCR was performed in triplicate with total uterine RNA (CLONTECH Laboratories, Inc., Palo Alto, CA) as a positive control. RNA expression levels were quantitated by comparing threshold cycles of the samples against the standard curve generated by positive controls. The experiment was valid if the negative RT controls had fluorescence intensity signals that were 100-fold less than experimental samples and if the size of the PCR products was verified by gel electrophoresis. RNA expression levels from the ovariectomized/injection model were normalized to Rpl-7 as a control (40), and samples from d 2.5 p.c. and d 3.5 p.c. were normalized to 18S.
RNA Sample Preparation for P4 Time Series
The mice used for the wild-type P4 time series were of 129/SvImJ background (The Jackson Laboratory, Bar Harbor, ME). In the wild-type P4 time series, total uterine RNA was extracted from ovariectomized mice at 0 (no injection), 1, 3, 6, 9, 12, 15, 18, and 24 h after P4 injection. Samples containing pooled total uterine RNA from at least four mice were used for each time point. The time course dataset was generated in two sets (0, 1, 3, 6, 9 h) and (0, 6, 12, 15, 18, 24 h). Samples in the same batch were processed in parallel at all steps, including ovariectomy, P4 injection, RNA collection, in vitro translation labeling, and chip hybridization. Time points of 0 h and 6 h were acquired in both batches to allow comparison between batches. The correlation between duplicates (at 0 h and 6 h) was much higher (r2 > 0.975) than that among samples within the same batch. Therefore, all time points were merged into one dataset for analysis.
SOM Analysis of Gene Expression in P4 Response Time Series
dChip expression levels from experiments using wild-type ovariectomized mice at nine time points (0, 1, 3, 6, 9, 12, 15, 18, and 24 h) after P4 injection were used, and only genes with at least a 2-fold change and absolute signal change of at least 75 U between any two time points were included in the SOM analysis (Genecluster, Whitehead Genome Center, Massachusetts Institute of Technology, Boston, MA) (15). Expression profiles for 1675 genes fulfilling these criteria were normalized before clustering so that the expression profile of each gene has mean 0 and variance 1 on the log scale. The number of nodes was incrementally increased until no further expression patterns emerged. A 3 x 6 geometry of nodes is depicted in the supplemental data published on the Endocrine Societys Journals Online web site, http://mend.endojournals.org (12). Clustering by SOM was also performed by using a 1.5-fold cutoff filter, with similar qualitative results.
Statistical Methods for Determining Functional Category Enrichment
A hypergeometric distribution was used to calculate the probability of observing the number of DRGs within each cluster in the time series. The probability P of observing at least k DRGs within a cluster of size (n) is:
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In Situ Hybridization and Immunohistochemistry
Uteri were cut into 46 mm pieces and flash frozen in Histo-Freeze (Fisher Scientific, Pittsburgh, PA). Frozen sections (11 µm) were mounted onto poly-L-lysine coated slides and fixed in cold 4% paraformaldehyde in PBS. Sections were prehybridized and hybridized at 45 C for 4 h in 50% formamide hybridization buffer containing the 35S-labeled antisense cRNA probe (specific activities 2 x 109 dpm/µg). After hybridization and washing, sections were incubated with ribonuclease A (20 µg/ml) at 37 C for 20 min. Ribonuclease A-resistant hybrids were detected by autoradiography using Kodak NTB-2 liquid emulsion (Eastman Kodak Co., Rochester, NY). Sections hybridized with the corresponding sense probe served as negative controls. Slides were poststained with hematoxylin and eosin. 35S-labeled riboprobes were generated using specific RNA polymerases. An IMAGE clone of p15 in pCMV-SPORT6 (IMAGE clone 3495097, Genbank accession no. BC002010) was purchased from Research Genetics, Inc. (Huntsville, AL) and confirmed by sequencing. Plasmid for the p57 riboprobe was a generous gift from Dr. Stephen Elledge.
For immunohistochemistry, uteri were cut into 46 mm pieces and flash frozen in Histo-Freeze (Fisher Scientific, Pittsburgh, PA). Frozen sections (12 µm) were mounted onto poly-L-lysine coated slides and fixed in Bouins fixative and washed in PBS. Immunostaining with the primary antibody (anti-CD4, H129.9, PharMingen, San Diego, CA) was performed using a Histostain kit from Zymed Laboratories, Inc. (South San Francisco, CA) following the manufacturers protocol.
Flow Cytometric Analysis of USCs and Splenocytes
USCs were isolated from groups of four to five wild-type and Hoxa-10 mutant mice on d 2.5 p.c. or d 3.5 p.c. as specified. In each group, uteri excluding the utero-oviductal junction were excised, trimmed of fat and mesentery, rinsed in PBS, flushed of embryos, pooled, and minced into fine fragments. Four rounds of 5-min incubation using collagenase type I (Sigma), mechanical disruption by pipetting, and 5-min sedimentation by gravity followed by removal of supernatant were performed in serum-free DMEM/F-12 (Life Technologies, Inc.) containing 1% penicillin-streptomycin (Sigma). Supernatant containing isolated USCs was passed through a 70-µm cell strainer [Falcon (BD Biosciences, Bedford, MA)] to remove cell clumps. Cell suspensions were centrifuged at 1400 rpm, 4 C, for 5 min, and USCs were resuspended in fluorescence-activated cell sorting (FACS) buffer (PBS, 0.5% BSA, 0.02% sodium azide) on ice.
Spleens were removed and pooled from the same groups of mice and kept in PBS with 5% fetal calf serum on ice. Splenocytes were prepared by homogenization of the spleen capsule using the plunger end of a syringe, passed through a 70-µm cell strainer, and suspended in PBS containing 5% fetal calf serum. Cells were pelleted by centrifugation (1800 rpm, 4 C, 5 min), resuspended in ACK buffer (0.15 M NH4Cl, 1.0 mM KHCO3, and 0.1 mM Na2EDTA) for 5 min to lyse red blood cells, repelleted by centrifugation, and resuspended in FACS buffer. A suspension of USCs or splenocytes (100 µl) was incubated with normal rabbit serum (PharMingen) at 1:50 dilution for 5 min on ice, followed by centrifugation and aspiration of supernatant. Cell pellets were resuspended in 50 µl of directly fluorochrome-conjugated monoclonal antibodies diluted in FACS buffer to a concentration of 0.1 mg/ml for 30 min in the dark on ice. Cells were washed twice in FACS buffer and analyzed on a FACScan (Becton Dickinson and Co.) with Cellquest software within 2 h of staining. The monoclonal antibodies used were (BD PharMingen): fluorescein isothiocyanate (FITC)-anti-
TCR (GL3), FITC-anti-TCR ß (H57597), FITC-anti-Pan-NK (DX5), FITC-anti-CD19 (1D3), FITC-anti-CD4 (H129.9), PE-anti-
TCR (GL3), PE-anti-TCR ß (H57597), PE-anti-Pan-NK (DX5), PE-anti-CD19 (1D3), and PE-anti-CD4 (H129.9).
Cell Proliferation
Four hours before the mice were killed, 100 µl (1 mg) of BrdU (BD Biosciences) was injected ip into each mouse. Cell staining was performed as above followed by cell fixing, permeabilization, deoxyribonuclease treatment, and BrdU staining using the BrdU Flow Kit (BD Biosciences) according to the manufacturers protocol. Fixed and stained cells were kept in FACS buffer in 4 C overnight and analyzed by FACS the next morning.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Present Address: Department of Obstetrics and Gynecology, Columbia University College of Physicians and Surgeons, New York, NY 10032.
Abbreviations: BrdU, Bromodeoxyuridine; cdk, cyclin-dependent kinase; CKI, cyclin-dependent kinase inhibitor; DRG, differentially regulated gene; E2, estradiol; EST, expressed sequence tag; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; P4, progesterone; p.c., post coital; PR, progesterone receptor; PRKO, PR knockout; RT, room temperature; SAM, statistical analysis of microarrays; SOM, self-organizing map; USCs, uterine stromal cells.
Received for publication August 20, 2002. Accepted for publication December 16, 2002.
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REFERENCES |
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