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Endocrinology, doi:10.1210/en.2007-0122
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Endocrinology Vol. 148, No. 9 4238-4250
Copyright © 2007 by The Endocrine Society

The p160 Steroid Receptor Coactivator 2, SRC-2, Regulates Murine Endometrial Function and Regulates Progesterone-Independent and -Dependent Gene Expression

Jae-Wook Jeong, Kevin Y. Lee, Sang Jun Han, Bruce J. Aronow, John P. Lydon, Bert W. O’Malley and Francesco J. DeMayo

Department of Molecular and Cellular Biology (J.-W.J., K.Y.L., S.J.H., J.P.L., B.W.O., F.J.D.), Baylor College of Medicine, Houston, Texas 77030; and Division of Biomedical Informatics (B.J.A.), Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229

Address all correspondence and requests for reprints to: Francesco J. DeMayo, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030. E-mail: fdemayo{at}bcm.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of the p160 steroid receptor coactivator 2 (SRC-2) in the regulation of uterine function and progesterone (P4) signaling was investigated by determining the expression pattern of SRC-2 in the murine uterus during pregnancy and the impact of SRC-2 ablation on uterine function and global uterine gene expression in response to progesterone. SRC-2 is expressed in the endometrial luminal and glandular epithelium from pregnancy d 0.5. SRC-2 is then expressed in the endometrial stroma on pregnancy d 2.5–3.5. Once the embryo is implanted, SRC-2 is expressed in the endometrial stromal cells in the secondary decidual zone. This compartmental expression of SRC-2 can be mimicked by treatment of ovariectomized mice with estrogen and P4. Ablation of SRC-2 in the uterus resulted in a significant reduction in the ability of the uterus to undergo a hormonally induced decidual reaction. Microarray analysis of RNA from uteri of wild-type and SRC-2–/– mice treated with vehicle or P4 showed that SRC-2 was involved in the ability of progesterone to repress specific genes. This microarray analysis also revealed that the uteri of SRC-2–/– mice showed alterations in genes involved in estrogen receptor, Wnt, and bone morphogenetic protein signaling. This analysis indicates that SRC-2 regulates uterine function by modulating the regulation of developmentally important signaling molecules and the ability of P4 to repress specific genes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ABILITY OF the uterus to support pregnancy is a steroid hormone-regulated process, largely due to the combined actions of both estrogen (E2) and progesterone (P4). Steroid hormones coordinate the progression of changes in the uterus that support embryo implantation and fetal development. In the mouse, pregnancy lasts 19–21 d with embryo implantation occurring on d 4.5 (d 0.5 is the morning after ovulation and copulation). The preovulatory E2 surge stimulates uterine epithelial cell proliferation during early pregnancy. As pregnancy progresses, P4 secreted from the newly formed corpus luteum initiates uterine stromal cell proliferation (d 2.5). During this period of P4 priming in combination with a secondary preimplantation and ovarian E2 secretion, uterine stromal proliferation and differentiation occur, rendering the uterus receptive for implantation (1, 2, 3, 4). At the site of blastocyst attachment on d 4.5 of pregnancy, the endometrial stromal cells undergo the decidual reaction, in which they proliferate and differentiate to form morphologically distinct decidual cells. The ability of the endometrial stromal cells to differentiate into decidual cells is a process that is dependent upon steroid hormone stimulation (5). Understanding the molecular mechanisms of action of E2 and P4 allows the definition of the processes regulating uterine function.

E2 and P4 exert their effects in the uterus primarily through modulation of gene transcription by their cognate nuclear receptors (6, 7). E2 acts through one of two estrogen receptors (ERs), ER-{alpha} or ER-ß, which are encoded by two separate genes (8, 9). P4 acts through the progesterone receptor (PR), which consists of two isoforms, PR-A and PR-B. These two isoforms arise from differential transcription of the PR gene (10, 11, 12). Gene ablation studies have demonstrated that ER-{alpha} and PR-A are primary regulators of uterine function (13, 14). These studies also show that whereas ER-{alpha} plays a permissive role in the regulation of uterine function, PR-A has been demonstrated to be the receptor that is critical for the uterus to support pregnancy (15). These receptors do not act alone in the regulation of gene transcription but are aided by coactivator proteins.

Transcriptional coactivator proteins facilitate steroid receptor regulation of gene transcription by executing a diverse number of processes from chromatin remodeling, RNA processing, and receptor degradation (16). One family of coactivators that functions as signaling intermediates between the nuclear receptor and the basal transcriptional machinery to enhance transcriptional activation is the p160, steroid receptor coactivator proteins (SRCs). The SRC family is composed of three distinct but functionally and structurally related members: SRC-1/NcoA1 (17, 18), SRC-2/TIF2/GRIP1 (19, 20), and SRC-3/RAC3/ACTR/pCIP/AIB1/TRAM1 (21, 22). The SRC family members enhance the transcriptional activity of a variety of nuclear receptors, including ER-{alpha}, ER-ß, and PR (17, 23, 24, 25, 26) and are expressed in a variety of hormone-responsive tissues including uterus, brain, prostate, and breast (18, 27, 28, 29, 30). A mouse model with disruption of the SRC-1 gene exhibited a phenotype of reduced steroid sensitivity. Although female SRC-1–/– mice were fertile, this reduced steroid sensitivity in the uterus was demonstrated by a decrease in the ability of the endometrial stromal cells to undergo a decidual transformation (18). SRC-2 female mice exhibit hypofertility due to a placental hypoplasia (31). Conditional ablation of SRC-2 using the PR-Cre mouse model also shows reduced steroid sensitivity for decidual transformation in the uterus as observed with the ablation of SRC-1 (32). SRC-3 null mice have disrupted female reproductive function, including delayed puberty and prolonged estrous cycles compared with their wild-type littermates; however, the impact of SRC-3 ablation on uterine function has not been investigated (21). The phenotype of the SRC family knockout mice indicates that these coregulators play a necessary role in coordinating steroid hormone regulation of normal reproductive uterine function.

Here, we investigate the temporal and spatial expression and role of all three members of the SRC family in the uterus during pregnancy. This analysis demonstrates that SRC-2 is coexpressed with SRC-1 in the uterus during pregnancy and that ablation of SRC-2 impacts the uterine response to steroid hormone induction of the decidual reaction. Furthermore, microarray analysis of the uteri of the SRC-2 null mice shows that SRC-2 is involved in the modulation of estrogen, Wnt, and bone morphogenetic protein (BMP) growth factor signaling pathways as well as in the ability of P4 to repress the expression of specific genes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tissue preparation
Mice were maintained in the designated animal care facility at the Baylor College of Medicine according to the Institutional Guideline for the Care and Use of Laboratory Animals. Groups of wild-type mice at 6 wk of age were ovariectomized. Two weeks later, ovariectomized mice were injected with vehicle (sesame oil), P4 (1 mg/mouse in 100 µl sesame oil), E2 (0.1 µg/mouse in 100 µl sesame oil), or E2 plus P4 for 4, 16, or 40 h (Sigma-Aldrich, St. Louis, MO). The injections were repeated every 12 h for the 16- and 40-h samples. The mice were anesthetized with Avertin (2,2,-tribromoethyl alcohol; Sigma-Aldrich) and killed by cervical dislocation at 4 h after the last injection to collect the uteri. Different days of pregnant uterine samples were obtained by the mating of wild-type mice, and the day that a vaginal plug was observed was considered as d 0.5 of pregnancy. Uterine tissues were flash frozen at the time of dissection or fixed with 4% paraformaldehyde (vol/vol) and paraffin embedded.

Hormonally induced decidual reaction
Ovariectomized SRC-1–/– (18), SRC-2–/– (31, 33), SRC-3–/– (21), and wild-type mice were treated with three daily injections of 100 ng E2 per mouse (n = 5 per genotype). After 2 d rest, mice were then treated with three daily injections of 1 mg P4 and 6.7 ng E2 per mouse by sc injection. The uteri were mechanically stimulated by a scratch of the antimesometrial lumen 6 h after the last injection. Mice were given daily sc injections of 1 mg P4 and 6.7 ng E2 per mouse for 5 d after stimulation to follow the induction of the uterine decidual response.

Quantitative real-time PCR
Total RNA was extracted from the uterine tissues using the QIAGEN (Valencia, CA) RNeasy total RNA isolation kit. Expression levels of mRNA were measured by real-time RT-PCR TaqMan analysis using the ABI Prism 7700 Sequence Detector System according to the manufacturer’s instructions (PE Applied Biosystems, Foster City, CA). For SRC-1, SRC-2, SRC-3, ER-{alpha}, Nrip1, Pcaf, Pparbp, Cdh2, Ppp2r2b, Sox7, Bmp2, Hoxa10, Bmp5, Wnt7a, Wif1, and 18S RNA, prevalidated probes and primers were purchased from Applied Biosystems. RT-PCRs were performed using One-step RT-PCR Universal Master Mix reagent and TaqMan Gene Expression Assays (Applied Biosystems) according to the manufacturer’s instructions. Standard curves were generated by serial dilution of a preparation of total RNA isolated from whole-mouse uterus. All real-time PCR was done by using the three independent RNA sets. All mRNA quantities were normalized against 18S RNA using ABI rRNA control reagents.

Immunohistochemistry
Uteri were fixed overnight in 4% paraformaldehyde (vol/vol), followed by thorough washing in 70% ethanol, and tissues were processed, embedded in paraffin, and sectioned. Uterine sections from paraffin-embedded tissue were cut at 5 µm and mounted on silane-coated slides, deparaffinized, and rehydrated in a graded alcohol series. Sections were preincubated with 10% normal rabbit or goat serum in PBS (pH 7.5) and then incubated with 1:500 anti-SRC-1 antibody or 1:1000 anti-SRC-2 antibody in 10% normal serum in PBS (pH 7.5). Polyclonal antibody for SRC-1 was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-SRC-2 antibody was kindly provided by Jun Qin of Baylor College of Medicine. On the following day, sections were washed in PBS and incubated with biotinylated secondary antibody (5 µl/ml; Vector Laboratories, Burlingame, CA) for 1 h at room temperature. Immunoreactivity was detected by using the Vectastain Elite ABC kit (Vector); the immunoreactivity was visualized as brown staining.

ß-Galactosidase activity staining
SRC-3 expression in the uterus was assayed using the SRC-3+/– mice. In the processes of ablating the SRC-3 gene, a promoter-free lacZ (ß-galactosidase) reporter gene was inserted in-frame to the N-terminal coding region (21). This lacZ knock-in approach allowed us to assay for SRC-3 expression using ß-galactosidase staining. Frozen uterine sections were fixed in ice-cold 4% paraformaldehyde (vol/vol) for 5 min. Fixed sections were subsequently rinsed in PBS. Uterine sections were then immersed in lacZ staining solution [2 mM MgCl2, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6·3H2O, 0.02% Nonidet P-40, 0.01% deoxycholate, and 1 mg/ml 4-chloro-5-bromo-3-indolyl-ß-D-galactopyranoside in 100 mM phosphate buffer, pH 8.0] at room temperature in the dark for at least 2 h and up to 16 h depending upon the degree of color reaction (Sigma-Aldrich).

Microarray analysis
Microarray analyses were performed as described previously (34). Eighteen SRC-2–/– and 18 wild-type mice were ovariectomized at 6 wk of age. Two weeks later, ovariectomized mice were injected with either vehicle (sesame oil) or P4 (in sesame oil) (Sigma-Aldrich) (1 mg/mouse in 100 µl) for 4 h (n = 3 per genotype per treatment). Groups of mice were killed 4 h after the injection. Each treatment consisted of nine SRC-2–/– and nine wild-type mice. Total RNA was extracted from the uterine tissues using the QIAGEN RNeasy total RNA isolation kit. The RNA was pooled from the uteri of three mice per genotype and treatment. All RNA samples were analyzed with a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA) before microarray hybridization. The fragmented, labeled cRNA (15 µg) was hybridized to Affymetrix Mouse Genome 430 2.0 Arrays (Affymetrix, Santa Clara, CA). Expression analysis of all replicate microarray experiments was performed with GeneSpring 7.2 (Agilent Technologies) using the absolute analysis data generated by Microarray Suite version 5.0 software (MAS 5.0; Affymetrix). Differential expression was defined as those transcripts that had a difference of 1.5-fold. Statistical analysis of the data was performed with the GeneSpring software version 7.2. A one-way ANOVA Welch t test for unpaired comparison of the two groups was applied. We set a P value of <0.05 to define a set of significantly up- and down-regulated genes. Differentially regulated genes identified in microarray analyses were analyzed using Ingenuity Pathways Analysis (Ingenuity Systems, Mountain View, CA). Analyses were conducted based on 1.5-fold up- or down-regulated genes (P < 0.05). A data set containing gene identifiers and corresponding expression values was uploaded into the application. Each gene identifier was mapped to its corresponding gene object in the Ingenuity Pathways Knowledge Base. Canonical pathways analyses identified the pathways from the Ingenuity Pathways Analysis library of canonical pathways that were most significant to the data set.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The expression profile of SRC-1, SRC-2, and SRC-3 in the pregnant mouse uterus
The temporal and spatial expression profile of SRC-1 and SRC-2 was investigated in the mouse uterus during early pregnancy by immunohistochemistry. The expression pattern of SRC-1 and SRC-2 in the pregnant mouse uterus followed a similar pattern of expression. As shown in Fig. 1Go, SRC-1 and SRC-2 were expressed in the luminal and glandular epithelium on d 0.5 (Fig. 1Go, a and b). The expression of SRC-1 and SRC-2 was transiently expressed in the endometrial stroma on d 2.5–3.5 of pregnancy (Fig. 1Go, d, e, g, and h), and then the endometrial stromal signal decreased on d 4.5 (Fig. 1Go, j and k) during the window of uterine receptivity. Once embryo implantation was initiated and stromal cells started to differentiate into decidual cells on d 5.5–6.5, strong SRC-1and SRC-2 staining was detected in the secondary decidual zone (Fig. 1Go, m, n, p, and q). Interestingly, these results indicated a similar pattern of expression for PR in the stromal cells during decidualization (35). These results suggest that SRC-1 and SRC-2 have an important function of PR-dependent decidualization during early pregnancy.


Figure 1
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  		FIG. 1. Localization of SRC-1, SRC-2, and SRC-3 expression in mouse uteri during early pregnancy. Immunohistochemistry for the SRC-1 (a, d, g, j, m, and p) and SRC-2 (b, e, h, k, n, and q) and X-gal staining for SRC-3 (c, f, i, l, o, and r) were performed on mouse uteri during early pregnancy. The mice were killed to harvest uteri at the indicated times of early pregnancy. Nuclei are lightly counterstained with methyl green or nuclear fast red, respectively.

 
SRC-3 expression in the uterus was assayed using the SRC-3+/– mice. In the processes of ablating the SRC-3 gene, a promoter-free lacZ (ß-galactosidase) reporter gene was inserted in-frame to the N-terminal coding region (21). This lacZ knock-in approach allowed us to assay for SRC-3 expression by using X-gal staining. SRC-3 expression could be detected only in the myometrium and vasculature of the endometrium before pregnancy d 5.5. (Fig. 1Go, c, f, i, and l). Interestingly, unlike SRC-1 and -2, the expression of SRC-3 was not detected in the endometrial stromal cells until after implantation in the primary decidual zone on d 5.5 of pregnancy (Fig. 1Go, o and r). These results suggest that SRC-1 and SRC-2 have a similar pattern of expression during pregnancy that is distinct from the pattern of expression of SRC-3. The pattern of expression of SRC-1 and SRC-2 in the uterus indicates that these proteins likely play a role in the preparation of the uterus for embryo implantation and that these proteins play cooperative roles in regulating steroid hormone action in the murine uterus.

Steroid hormone regulation of SRC-1 and SRC-2 in the murine uterus
The above analysis demonstrates that the expression patterns of the SRC-1 and SRC-2 fluctuate in a compartmental and stage-specific pattern in the murine uterus; this pattern is similar to the expression pattern of the PR during early pregnancy. Because PR expression in the uterus is mediated by E2 and P4, we next determined the effect of steroid hormone stimulation on the levels of SRC-1 and SRC-2 in the uterus. Ovariectomized mice were injected with vehicle (sesame oil), P4 (1 mg/mouse in 100 µl sesame oil), E2 (0.1 µg/mouse in 100 µl sesame oil), or E2 plus P4 with repeat injections every 12 h (Fig. 2AGo). Immunohistochemistry was used to determine the effect of E2 and P4 on SRC-1 and SRC-2 expression. In the control ovariectomized mouse uterus, strong expression of the SRC-1 and SRC-2 protein was observed in luminal epithelium and glandular epithelium, whereas weak staining of stromal cells in vehicle-treated uterus was observed (Fig. 2BGo, a–c, and 2CGo, a–c, respectively). SRC-1 and SRC-2 protein in luminal and glandular epithelium in P4-treated uteri for 16 and 40 h continued to be expressed, whereas the stromal staining increased (Fig. 2BGo, d–f, and 2CGo, d–f, respectively). E2 treatment influences the localization of SRC-1 and SRC-2 protein in the mouse uterus (Fig. 2BGo, g–i, and 2CGo, g–i, respectively). The staining of the SRC-1 and SRC-2 in the stromal cells was increased by E2 treatments. E2 plus P4-treated uteri had the same expression patterns in both P4 and E2 treatment (Fig. 2BGo, j–l, and 2CGo, j–l). These results show that P4 and/or E2 increased the expression of SRC-1 and SRC-2 in the stromal cells. Sections of SRC-1–/– and SRC-2–/– uteri served as negative controls for the immunohistochemical analyses and did not exhibit any specific immunostaining (data not shown). Our results suggest that P4 and/or E2 induce the expression of SRC-1 and SRC-2 in stromal cells.


Figure 2
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  		FIG. 2. Localization of SRC-1 and SRC-2 expression in the ovariectomized mouse uteri. Ovariectomized mice were treated with P4, E2, E2 plus P4, or vehicles with repeat injections every 12 h. A, Four hours after the last injection, the mice were killed to harvest uteri at 4, 16, and 40 h; B and C, immunohistochemical analysis of SRC-1 (B) and SRC-2 (C) expression in the ovariectomized mouse uteri after steroid hormone treatment: a–c, uterine samples from the ovariectomized mice given sesame oil as control (Veh.); d–f, uterine samples from the ovariectomized mice given P4; g–i, uterine samples from the ovariectomized mice given E2; j–l, uterine samples from the ovariectomized mice given both E2 and P4. Nuclei are lightly counterstained with methyl green.

 
The impact of SRC-2 ablation on uterine decidualization
Previous analysis demonstrated that ablation of SRC-1 reduces the uterine response to steroid hormone stimulation of the uterine stromal decidual response (18). Because SRC-2 is expressed in a similar pattern as SRC-1 (see above) and transient transfection analysis has shown that the SRCs have similar activity, we next investigated the role of SRC-2 in regulation of mouse uterine function. This was accomplished by assaying the extent of the uteri of SRC-2–/– mice during a decidual reaction in response to steroid hormone stimulation. Although the expression pattern of SRC-3 was distinct from SRC-1 and SRC-2, this analysis was extended to SRC-3–/– mice to determine its role in the regulation of uterine function. Therefore, this analysis would determine the extent to which all individual members of the SRC family regulate uterine function.

Ovariectomized SRC-2–/– (n = 5), SRC-3–/– (n = 5), and littermate control SRC-2+/+ (n = 5) and SRC-3+/+ (n = 5) mice were treated with E2 and P4 to mimic pregnancy and the uterus was stimulated (by a scratch) to mimic the signaling of the embryo at implantation and to induce decidualization (see Materials and Methods). SRC-2–/– mice displayed 60.55% reduction in the decidual response, which is a level that is significant when compared with SRC-2+/+ mice (Fig. 3AGo, a and b). This reduction was confirmed by histological analysis of stromal differentiation (Fig. 3AGo, e and f). Analysis of the expression of Hoxa10 and Bmp2, regulators of decidualization, in the wild-type and SRC2–/– decidual horn was assayed by real-time RT-PCR (Fig. 3AGo, g). No significant difference in the mRNA for Hoxa10 and Bmp2 was observed in these samples. Although there appears to be an increase in Bmp2 mRNA, this change was not statistically significant. This indicates that although decidualization did occur at the morphological and molecular level in the SRC-2–/– uteri and a difference in the extent of decidualization at the morphological level could be detected, changes at the molecular level could not be detected with these two regulators of decidualization. Analysis of all of the littermate wild-type controls and the SRC-3–/– mice showed a typical increase in uterine weight in response to decidualization stimuli (Fig. 3AGo, c and d). These results demonstrate that, similar to the SRC1–/– mice, SRC-2–/– mice have a partial defect in responding to steroid hormone induction of uterine decidualization, whereas ablation of SRC-3 does not have any impact on the decidual response.


Figure 3
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  		FIG. 3. Decidual defect of the SRC-2–/– mice. A, Decidualization response of SRC-2–/– and SRC-3–/– mice; a and c, artificially induced decidualized uteri in SRC-2–/– (a), SRC-3–/– (c), and wild-type (WT) mice (bar, 1 cm); b and d, quantitative measuring of decidual response in the SRC-2–/– (b), SRC-3–/– (d), and wild-type mice. Each uterine horn was weighted on d 5 after streaking stimulation in SRC-2–/– and wild-type mice. The results represent the mean ± SEM of three animals. **, P < 0.01. e and f, Hematoxylin and eosin staining of the artificially induced decidualized uteri in wild-type (e) and SRC-2–/– (f) mice; g, real-time RT-PCR analysis of the Hoxa10 and Bmp2 mRNAs in stimulated SRC-2–/– and wild-type mice uteri on d 5 after artificially induced decidualization. B, Expression of the SRC-1, SRC-2, and SRC-3 in artificially induced decidualized uteri; a–c, real-time RT-PCR analysis of the SRC-1 (a), SRC-2 (b), and SRC-3 (c) mRNAs in nonstimulated and stimulated SRC-1–/–, SRC-2–/–, SRC-3–/–, and wild-type mice uteri on d 5 after artificially induced decidualization. The results represent the mean ± SEM of three independent RNA sets. **, P < 0.01.

 
Next, the impact of the ablation of each SRC member on the expression of the remaining SRC family members in the uterus during the decidual response was assayed. The mRNA levels of SRC-1, SRC-2, and SRC-3 in the hormonally induced decidualized uteri of SRC-1–/–, SRC-2–/–, SRC-3–/–, and wild-type mice was assayed by real-time RT-PCR (Fig. 3BGo). SRC-1, SRC-2, and SRC-3 mRNAs were significantly increased in the wild-type stimulated horn compared with the control horn (1.99-, 1.90-, and 2.69-fold, respectively). The expressions of SRC-1 and SRC-2 were increased in the stimulated uterine horn of the SRC-3–/– compared with the control horn (2.21- and 2.53-fold, respectively). This is not surprising because SRC-3 ablation does not impact the murine decidual response. However, induction of SRC-2 and SRC-3 mRNAs was not detected in the stimulated uterine horn of the SRC-1–/– mice compared with the control horn. A similar pattern with SRC-1–/– mice was observed in the SRC-2–/– mice. The expression levels of SRC-1 and SRC-3 mRNAs were not changed in the stimulated uterine horn of the SRC-2–/– mice compared with the control horn. These results suggest that SRC-1 and SRC-2 play important roles in decidualization and that each coactivator does not compensate for the loss of another member of the SRC family during decidualization in the murine uterus.

Identification of SRC-2 and P4 target genes in the murine uterus
P4 is a major steroid hormone for regulation of decidualization in the uterus, and ablation of SRC-2 dampens this P4-regulated process. Consequently, oligonucleotide microarray analysis was used to identify the genes in the mouse uterus that are significantly changed by SRC-2 ablation in the ovariectomized mice and in the response of the uterus to acute P4 stimulation. Ovariectomized SRC-2–/– and wild-type mice were treated with either P4 or vehicle (sesame oil) for 4 h. Pooled uterine total RNAs from three mice of each group were hybridized to Affymetrix Mouse Genome 430 2.0 Arrays. The experiments were carried out in triplicate using 12 Mouse Genome 430 2.0 Arrays. As generally adopted for oligonucleotide microarray profile analysis, we applied a threshold of a 1.5-fold change in expression level (P < 0.05). Using this experimental design, we used three physiologically relevant comparisons to identify differentially expressed genes. The three comparisons and the number of significantly differentially regulated genes in each comparison are listed in Table 1Go. Comparison 1 consisted of analyzing differential gene expression of wild-type mice treated with vehicle vs. wild-type mice treated with P4. Comparison 2 (wild-type-vehicle vs. SRC-2–/–-vehicle) identified differentially expressed genes between wild-type and SRC-2–/– mice treated with vehicle. This comparison identifies genes whose expression was due to the developmental consequences of ablation of the SRC-2. Comparison 3 identified differentially expressed genes between wild-type and SRC-2–/– mice treated with P4. This comparison identified genes regulated by SRC-2 in the presence of P4. All of the differentially regulated genes for each comparison are listed in supplemental Tables 1, 2, and 3, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org. The overall impact of P4 in the wild-type mice was that the number of genes with increased gene expression was greater than that of genes with decreased expression when compared with wild-type mice treated with vehicle. However, the impact of SRC-2 gene ablation leads to an overall down-regulation of gene expression, and the impact of P4 in the SRC-2–/– mice was consistent with overall down-regulation of gene expression.


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  		TABLE 1. Number of the significantly increased and decreased uterine genes in three comparisons of microarray analysis

 
To categorize the P4- and SRC-2-regulated genes into specific functional groups, the significantly increased and decreased genes were functionally categorized by gene ontology terms. The result of the sorting of the genes that filtered from the three comparisons is shown in Fig. 4Go, A–C, respectively. Transcription factors, transport proteins, signal transduction, cell growth, and enzyme genes were increased by acute treatment with P4 (Fig. 4AGo). As expected, these genes were decreased in SRC-2–/– treated with P4 (Fig. 4CGo). The impact of ablation of SRC-2 on gene expression in the unstimulated uterus also showed a decrease in similar groups of genes; however, unlike the other two comparisons, there was an increase in genes involved in immunity (Fig. 4BGo).


Figure 4
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  		FIG. 4. SRC-2- and P4-regulated genes in the mouse uteri: functional categorization, relationships, and cluster analysis of significantly regulated genes. Each gene expression level is given as the ratio relative to its corresponding level in wild-type (WT) vehicle (veh)-treated animals. Significantly regulated genes were identified based on ANOVA. A–C, Functional categorization of increased and decreased genes regulated by P4 (A), SRC-2 (B), and P4 and SRC-2 (C). Genes were annotated and assigned to various functional categories based on Affymetrix annotations. Increased genes are shown as red bars, and decreased genes are shown as blue bars. D, Venn diagrams demonstrating the relationship between genes modulated in the SRC-2–/– uterus in response to treatment with P4. Red circles indicate genes selected by vehicle-treated wild-type vs. P4-treated wild-type; green, by P4-treated SRC-2–/– vs. P4-treated wild-type; blue, vehicle-treated wild-type vs. vehicle-treated SRC-2–/–. The numbers, displayed within the intersections of the circles indicate the common genes by two comparisons. The number at the bottom right corner of the panel indicates total genes analyzed. E, Clustering analysis of SRC-2- and P4-regulated genes in the murine uteri. The pool of SRC-2- and P4-regulated genes (1011 genes) were clustered according to their gene expression patterns using the hierarchical-tree algorithm. The color code for the signal strength in the classification scheme is shown in the box at the bottom in which induced genes are indicated by red; repressed genes are indicated by green.

 
To identify trends in the changes in the gene expression of P4-SRC-2-regulated genes, stringent data filtering and clustering further allowed the grouping of the P4- and SRC-2-regulated genes into groups of biological relevance. To this end, we determined which differentially regulated genes were in common in our microarray results. The result is graphically represented in Fig. 4DGo. Interestingly, our results reveal a remarkably limited number of genes that overlap among the three groups.

All of P4- or SRC-2-regulated genes were analyzed by mathematical clustering using a hierarchical tree (Fig. 4EGo). P4 had the largest effect on uterine gene expression, and the large majority of this effect was not dependent upon SRC-2 (supplemental Tables 1 and 2). The largest SRC-2 effect consisted of a P4-independent loss of gene expression in the uterus (labeled repressed genes in SRC-2–/– mice). The next largest effect of the SRC-2–/– mice was a gain in gene expression among genes normally strongly repressed by P4. Most of these genes remained nonrepressible by P4; thus, both their basal and P4-regulated behaviors were dependent upon SRC-2. A smaller group of genes activated by P4 failed to be activated in the absence of SRC-2, but by and large, the bulk of the P4-activated genes did not require SRC-2. We next validated the ability of SRC-2 to impact the P4-dependent gene repression and then examined the role of P4 and SRC-2 to regulate the expression of members of the Wnt, BMP, and ER signaling pathways.

The repression of Calb1, Hmga2, and Cdkl1 by P4 in the murine uterus was significantly decreased by SRC-2 ablation
The overall goal of this microarray analysis was to identify genes that are coordinately regulated by SRC-2 and P4. We previously identified the genes whose uterine expression is markedly altered by P4 and PR (34). In that analysis, a limited number of genes were repressed by P4 treatment. However, in this analysis, we identified 107 genes as being down-regulated by P4. Of the genes regulated by P4, we have identified 21 genes whose repression is dampened by ablation of SRC-2 in the uterus of the mouse (Table 2Go). To validate the alteration of gene expression at the mRNA level that appeared on the microarray, real-time RT-PCR was performed. Expression of Calb1, Hmga2, and Cdkl1 was significantly decreased by P4 in the wild-type uteri (39.1, 24.4, and 18.0%, respectively). The repression of these genes was attenuated in the SRC-2–/– uteri treated with P4 (Fig. 5Go). Thus, these results were consistent with the microarray-based identification of these genes.


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  		TABLE 2. The list of uterine genes whose P4 regulation was affected by SRC-2 ablation

 

Figure 5
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  		FIG. 5. Validation of SRC-2- and P4-regulated genes in the murine uterus. Real-time RT-PCR analysis of the Calb1, Hmga2, and Cdkl1 in vehicle (Veh)- and P4-treated wild-type (WT) and SRC-2–/– mouse uteri. The results represent the mean ± SEM of three independent RNA sets. *, P < 0.05; **, P < 0.01.

 
P4 gene repression includes members of the Wnt signaling cascade
Wnt signaling-related genes Wnt7a (wingless-related MMTV integration site 7A), Cdh2 (cadherin 2), Ppp2r2b (protein phosphatase 2 (formerly 2A), regulatory subunit B (PR 52), ß-isoform), and Sox7 (SRY-box containing gene 7) were decreased –7.09-, –2.25-, –1.74-, and –4.33-fold in the wild-type mice treated with P4 compared with wild-type mice treated with vehicle, respectively (supplemental Table 1).

These gene expression profiles were validated in the different mice by real-time RT-PCR (Fig. 6Go). The expressions of Wnt7a and Sox7 were significantly suppressed (18.4 and 34.0%, respectively) in wild-type mice treated with P4, and the suppression was observed in the SRC-2–/– mice (48.4 and 62.7%, respectively). However, this suppression was decreased significantly in the SRC-2–/– mice treated with P4 compared with wild-type mice treated with vehicle. The expressions of Cdh2 and Ppp2r2b were significantly decreased in the wild-type mice (46.2 and 35.9%, respectively) and the SRC-2–/– mice (28.5 and 9.1%, respectively) treated with P4. The results were consistent with microarray identification of these genes as SRC-2 and P4 targets. Our results suggest that Wnt signaling-related genes (Wnt7a, Sox7, Cdh2, and Ppp2r2b) are down-regulated by P4 in the uterus of wild-type mice and that the repression of Wnt7a and Sox7 by P4 that is dependent upon SRC-2, in contrast to the repression of Cdh2 and Ppp2r2b by P4, is independent of SRC-2.


Figure 6
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  		FIG. 6. Down-regulation of Wnt signaling pathway genes by P4 in the uterus and validation of Wnt signaling pathway genes in the uterus by real-time RT-PCR analysis of the Wnt7a, Sox7, Cdh2, and Ppp2r2b in vehicle (Veh)- and P4-treated wild-type (WT) and SRC-2–/– mice uteri. The results represent the mean ± SEM of three independent RNA sets. *, P < 0.05; **, P < 0.01.

 
An additional gene member of the Wnt signaling pathway, Wif1 (Wnt inhibitory factor 1, WIF1) revealed altered expression in the uterus of SRC-2–/– mice compared with the wild-type mice. This microarray result was validated by real-time RT-PCR (Fig. 7AGo). The expression level of Wif1 was decreased in the uterus of SRC-2–/– mice compared with the wild-type mice. The expression of Wif1 mRNA was decreased in P4-treated wild-type and SRC-2–/– mice compared with vehicle-treated mice (15.4 and 50.0%, respectively). However, this repression was not observed in the PR knockout (PRKO) mice (Fig. 7AGo). The results suggest that the modulation of Wif1 by P4 was dependent on the presence of PR and not SRC-2. Interestingly, although Wif1 expression was repressed by P4 in a receptor-dependent manner, the basal level of Wif1 expression was decreased by the ablation of PR and SRC-2. This indicates that the unliganded PR is necessary for the basal level of expression of Wif1 and that the binding of ligand to the receptor results in the repression of Wif1 gene expression.


Figure 7
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  		FIG. 7. Regulation of Wif1, Bmp2, and Bmp5 expression by SRC-2, PR, and P4. A, Real-time RT-PCR analysis of the Wif1 in vehicle (Veh)- and P4-treated and SRC-2–/–, PRKO, and wild-type (WT) mouse uteri. The results represent the mean ± SEM of three independent RNA sets. *, P < 0.05; **, P < 0.01. B, Immunohistochemical analysis of WIF1 expression in the ovariectomized SRC-2–/–, PRKO, and wild-type mouse uteri after P4 or vehicle treatment. Ovariectomized mice were treated with P4 or vehicles. Four hours after the injection, the mice were killed to harvest uteri. Nuclei are lightly counterstained with methyl green. C, Regulation of Bmp2 and Bmp5 expression by SRC-2, PR, and P4. Real-time RT-PCR analysis of Bmp2 and Bmp5 was performed in vehicle- and P4-treated SRC-2–/–, PRKO, and wild-type mice uteri. D, Immunohistochemical analysis of BMP2 expression in the ovariectomized SRC-2–/–, PRKO, and wild-type mice uteri after P4 or vehicle treatment.

 
Next, we investigated cell type-specific expression of WIF1 in the uteri of ovariectomized wild-type mice treated with vehicle and wild-type, SRC-2–/–, and PRKO mice treated with P4. Immunohistochemical analysis showed that WIF1 protein is detected in the luminal epithelium and glandular epithelium in wild-type mice treated with vehicle (Fig. 7BGo). However, we could not detect any WIF1 protein in the uteri of wild-type, SRC-2–/–, or PRKO mice treated with P4. This analysis validated the real-time PCR analysis that repression of WIF1 expression is PR dependent upon P4 in the wild-type mouse uterus and that the basal expression of WIF1 is dependent upon PR and SRC-2.

Bmp2 and Bmp5 were suppressed by P4 and SRC-2 in the murine uterus
The BMP signaling-related genes Bmp2 and Bmp5 were decreased –1.558-fold and –1.541-fold, respectively, in the SRC-2–/– mice treated with P4 compared with wild-type mice treated with P4 (supplemental Table 3). The expressions of these gene profiles were validated in all mice by real-time RT-PCR (Fig. 7CGo). The expressions of Bmp2 and Bmp5 were significantly decreased by P4 in the wild-type mice compared with wild-type mice treated with vehicle (43.6 and 47.70%, respectively); differential expression of those genes was not observed in the SRC-2–/– uteri. However, this suppression was decreased in the SRC-2–/– and PRKO mice treated with P4 compared with SRC-2–/–, PRKO, and wild-type mice treated with vehicle. These results were consistent with the microarray data identification of these genes as SRC-2 and P4 targets and suggest that Bmp2 and Bmp5 are SRC-2- and PR-dependently repressed in the uterus of mouse.

Next, we investigated cell type-specific expression of BMP2 in the SRC-2–/–, PRKO, and wild-type mice treated with P4. Immunohistochemistry showed that BMP2 protein staining was observed in the luminal epithelium and glandular epithelium in the wild-type mice treated with vehicle (Fig. 7DGo). However, we could not detect staining in the wild-type mice treated with P4. As we expected, SRC-2–/– and PRKO mouse uteri treated with P4 had strong luminal epithelium and glandular epithelium staining. Interestingly, the localization of BMP2 in the luminal epithelium and glandular epithelium was matched exactly to the location of SRC-2 and SRC-1 in the wild-type mice treated with vehicle. The localization of SRC-2 and BMP2 supports our interpretation that BMP2 is a direct target gene of SRC-2.

The basal expression of ER signaling genes is decreased in the uterus of SRC-2–/– mice
Differentially regulated genes in the uterus of SRC-2–/– mice were analyzed using Ingenuity Pathways Analysis (https://analysis.ingenuity.com). The analyses were conducted based on 1.5-fold up- or down-regulated genes (P < 0.05). Canonical pathway analyses identified ER signaling pathways from the Ingenuity Pathways Analysis library of canonical pathways. The expressions of ER-{alpha}, Nrip1 (nuclear receptor interacting protein 1), Pcaf (p300/CBP-associated factor), and Pparbp (peroxisome proliferator-activated receptor binding protein) were –1.534, –1.678, –2.128, and –1.751, respectively, fold decreased in the SRC-2–/– mice compared with the wild-type mice in the absence of P4. We could not detect changes in these genes in the presence of P4. These results were validated by real-time RT-PCR, and all of the altered genes in the estrogen receptor signaling pathways were significantly decreased in the SRC-2–/– mice (Fig. 8AGo).


Figure 8
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  		FIG. 8. Alteration of ER signaling pathway in the uteri of SRC-2–/– mice. A, Validation of differently expressed genes in the ER signaling pathway. Real-time RT-PCR analysis of the ER-{alpha}, Nrp1, Pcaf, and Pparbp in wild-type (WT) and SRC-2–/– mice uteri. The results represent the mean ± SEM of three independent RNA sets. *, P < 0.05; **, P < 0.01. B, Immunohistochemical analysis of the ER-{alpha} expression in the uterus of SRC-2–/– and wild-type mice. Nuclei are lightly counterstained with methyl green.

 
Finally, we investigated cell type-specific expression of ER-{alpha} in the uteri of ovariectomized wild-type and SRC-2–/– mice. Immunohistochemical analysis showed that ER-{alpha} protein is detected in the luminal epithelium, glandular epithelium, stroma, and myometrium in the wild-type mice treated with vehicle (Fig. 8BGo). Expression of ER-{alpha} was significantly decreased in the luminal epithelium and myometrium of SRC-2–/– mice but not stromal and glandular epithelium.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The steroid receptors PR, ER-{alpha}, and ER-ß mediate the effects of P4 and E2 for implantation and pregnancy maintenance in the uterus (18, 21, 28, 36). In this study, we evaluated the temporal and spatial expression of the coactivators SRC-1, SRC-2, and SRC-3 in the murine uterus during early pregnancy. The uterine expression patterns of SRC-1 and SRC-2 are similar and are regulated by the steroid hormones P4 and/or E2 during early pregnancy. SRC-3 is not expressed in the endometrium of the preimplantation mouse. Interestingly, during the postimplantation period, SRC-3 expression is induced in the primary decidual zone, whereas SRC-1 and SRC-2 are expressed in the secondary decidual zone. The expression of SRC-1, SRC-2, and SRC-3 during uterine decidualization testifies to an important function of the SRCs in regulating uterine function during implantation. The differential expression of SRC-1 and SRC-2 vs. SRC-3 can be correlated with a functional difference in the role of these coregulatory proteins in the uterus. Like SRC-1, SRC-2 plays a role in the uterine decidual reaction, whereas SRC-3 appears not to play a significant role in this process. The endogenous physiological function of PR in distinct tissues is modulated by different steroid receptor coregulators. SRC-1 is a primary coactivator for PR in uterus, whereas SRC-3 is a primary coactivator for PR in breast (37, 38).

SRC family members have been shown to interact with and enhance the transcriptional activity of nuclear receptors, including PR. SRC-1 was isolated originally in a two-hybrid screen by using PR ligand-binding domain as bait (17). This interaction of SRC-1 with PR as well as its coactivating function is P4 dependent (39). The other members of the SRC family members, SRC-2 (19) and SRC-3 (40), also increase the transcriptional activity of a variety of nuclear receptors, including PR. Female PR null mice (PRKO) exhibit pleiotropic reproductive abnormalities including a defect of decidualization (41, 42). Here, we report that the SRC-2–/– mouse shows a decreased decidual reaction in response to E2 and P4 treatment as observed in the SRC-1–/– mouse (18) as well as the PRCre/+ SRC-2flox/flox mouse (32). In fact, the decreased decidual response in the SRC-2–/– mouse is similar to the PRCre/+ SRC-2flox/flox mouse (data not shown) and indicates that the action of SRC-2 in regulating the uterus hormone responsiveness is primarily endogenous to the uterus and not due to systemic effects. However, more analysis must be conducted between these two models to make this conclusion. Because the expression of SRC-1 and SRC-2 in the uterus exhibits similar expression patterns to PR expression during early pregnancy, and ablation of SRC-1 and SRC-2 show an alteration in the PR-regulated decidual response, these two coactivators appear to function to facilitate cooperatively the role of PR during early pregnancy.

The coexpression of SRC-1 and SRC-2 in the uterus raises the possibility that these coactivators could compensate for each other in part in the regulation of uterine function. SRC-1–/– mice have normal body size, and their hormonal target tissues, such as the uterus, prostate, testis, and mammary gland exhibited decreased growth and development in response to steroid hormones (18). Here, we show that SRC-2–/– mice, but not SRC-3–/– mice, show a decrease in the uterine response to P4 during the decidual reaction. The analysis of SRC-1/SRC-2 compound mutant mice showed that SRC-1 can partially compensate for the effect of a loss of SRC-2 in mouse survival and growth (33). Indeed, a 2-fold overexpression of SRC-2 mRNA was detected in certain tissues, such as brain and testis in SRC-1 null mutants (18). We induced artificial decidualization in the SRC-1–/– mice and measured the expression levels and spatial distributions of SRC-2 and SRC-3. However, our results showed that SRC-2 and SRC-3 do not compensate for SRC-1 loss for uterine decidualization; SRC-1 and SRC-3 also do not compensate for the decidual defect seen in SRC-2–/– mice by an increase in transcription of these genes. Interestingly, the expression level of other SRC members are decreased in the artificially induced decidual region of SRC-1–/– and SRC-2–/– mice. Therefore, the compensation that occurs in the ablation of SRC-1 or SRC-2 alone may reflect a partial functional redundancy or the possibility that these coactivators may regulate independent/cooperative gene pathways. This was validated by showing that conditional ablation of SRC-2 in a SRC-1–/– mouse shows complete loss of the decidual response (32). We postulated that the identification of the regulatory pathways mediated by SRC-1 and SRC-2 would shed light on how these proteins mediate steroid responsiveness in the uterus. In fact, the microarray analysis of the uteri of SRC-2–/– mice did identify distinct pathways regulated by this coactivator.

We have identified 503 P4-regulated genes compared with our previous microarray analysis, which resulted in the identification of 90 P4-regulated genes (34). Most of the genes that were induced by P4 in the latest array (396 genes) were previously identified by microarray analysis as P4-stimulated genes using the Affymetrix Murine Genome U74Av2 Array (supplemental Table 1) (34). In addition, using the Affymetrix Mouse Genome U430 2.0 Array, a significant number of genes were identified as being repressed by P4 (107 genes; supplemental Table 1). The design of our microarray analysis was to determine the role of SRC-2 in the regulation of PR activity. This analysis identified PR-dependent as well as -independent genes whose expression was altered by SRC-2 ablation. In our investigation of the role of SRC-2 in uterine gene expression, the majority of genes altered by SRC-2 ablation did not affect the response of those genes to modulation by P4 (370 genes; supplemental Table 2). The limited impact of SRC-2 ablation on P4-regulated genes may be due to redundancy of SRC-1 for SRC-2 in the mediation of PR signaling because ablation of both genes in the uterus is required for loss of PR signaling. Therefore, this analysis identified gene pathways in which SRC-2 expression was required and may be independent of SRC-1 action. This was validated by showing that conditional ablation of SRC-2 in a SRC-1–/– mouse shows complete loss of the decidual response (32).

We found that ER signaling pathway-related genes were significantly decreased in the uterus of SRC-2–/– mice compared with wild-type mice in the absence of ligand. ER-{alpha}, Nrip1, Pcaf, and Pparbp gene expression was all significantly reduced. However, this did not result in the significant reduction in the expression of E2 target genes because SRC-1 and SRC-2 enhance ER-{alpha}- and PR-mediated gene transcription (17, 19). ER-{alpha} recruits SRC-1 and SRC-3 strictly in a hormone-dependent manner, whereas a ligand-independent interaction was observed with the receptor interaction domain of SRC-2 (43). The impact of the reduction in ER-2 and the loss of SRC-2 may have resulted in the mild attenuation of ligand-dependent and -independent gene expression that may not have significantly impacted one pathway but resulted in an overall dampening of decidual reaction in response to E2 and P4 stimulation. However, the alteration of ER signaling genes did not change their expression in the presence of P4. This may indicate a developmental reprogramming of the uterus due to SRC-2 ablation that resulted in a dampened response of the uterus to hormonal induction of the decidual reaction. Evidence for this is given by the alteration of BMP and Wnt pathways altered in the SRC-2–/– mouse that may have resulted in the developmental reprogramming of the uterus.

We identified BMP and Wnt signaling genes as SRC-2- and P4-regulated genes. Bmp2, Bmp5, and Wnt7a were P4-dependently repressed in the murine uterus, and this repression is dependent upon SRC-2 and PR in the uterus. BMPs have been shown to be induced by Wnt signaling in the neurons (44). Therefore, cooperation between Wnt and BMP signaling also appears to play an important role in reproductive function. Theca cells are responsive to BMPs in terms of steroidogenesis and proliferation; BMP2 and BMP6 act to suppress P4 synthesis by theca cells in culture (45, 46). We observed repression of Bmp2 expression by acute P4 treatment but not chronic treatment in the uterus of mouse in the uterine epithelial compartments. This repression was observed on d 2.5 of pregnancy, the time point when P4 is increased during early pregnancy in the uterus of the mouse. However, BMP2 and SRC-2 were highly expressed in the secondary decidual zone in the uterus. This would indicate that the regulation of BMP2 by P4 and SRC-2 may be different in the epithelial and stromal compartments. Chronic P4 treatment, as observed in pregnancy, may shift BMP2 expression from the epithelium to the decidual region. This change in compartmental expression and regulation during pregnancy may indicate changes in receptor expression patterns as well as changes in receptor-coactivator activity due to the integration of growth factor signaling between the epithelium and stroma during pregnancy. This correlation of expression of BMP2, SRC-2, and PR supports that BMP2 plays an important function in decidualization because it is a SRC-2 target gene in the uterus of the mouse. Wnt7a coordinates a variety of cell and developmental pathways that guide postnatal uterine growth and hormonal responses, and disruption of these pathways leads to cell death (47). Wnt7a mutant uteri do not form glands; the oviduct lacks a clear demarcation from the anterior uterus and acquires several cellular and molecular characteristics of the uterine horn (48, 49). Our results of SRC-2- and PR-dependent regulation in conjunction with the phenotype of Wnt7a–/– mice support the importance of Wnt7a in the uterus of the mouse.

One of the more surprising findings from this study is that SRC-2 aids in the P4 repression of gene expression. SRC-2 has been identified as a coactivator of gene expression (19, 24) and, therefore, is thought to aid PR primarily in the activation of gene transcription. SRC-2 is also required for transcriptional repression and acts as a corepressor for ER and glucocorticoid receptor (50, 51, 52). Interestingly, the microarray findings indicate that dependent upon the specific endocrine context, P4, through its receptor, represses the expression of specific genes, and this repression is dependent upon the presence of SRC-2. This finding corroborates previous findings that SRC-2 is involved in transcriptional repression as observed in the case of ER and glucocorticoid receptor gene repression and extends these findings to PR-repressed genes (50, 51, 52). However, this analysis does not prove that SRC-2 directly aids PR in gene repression. Because the SRC-2–/– mouse is a traditional gene knockout and SRC-2 expression has been ablated during development, the loss of additional factors necessary for mediating P4 repression of genes is likely. Identification of these factors should further our understanding of the mechanism by which P4 represses gene expression.

This microarray analysis identified genes in which the ablation of SRC-2 impacted the P4 repression of genes in ovariectomized mice given a pharmacological dose of P4. The genes whose repression by P4 is altered by SRC-2 ablation reflect cell cycle, immunity, structural proteins, transcription factors, and transport proteins. Investigation of the expression pattern of these genes during natural pregnancy combined with functional analysis of the role of these genes in the establishment and maintenance of pregnancy will determine the significance of the SRC-2 regulation of these genes during pregnancy.

In conclusion, we have demonstrated the individual roles of the nuclear receptor coactivators SRC-1, SRC-2, and SRC-3 for production of mRNA and protein expression in the decidua during pregnancy. The SRC-2-dependently regulated target genes have been identified by microarray analysis. ER-{alpha}, BMP, and Wnt signaling pathways as well as the P4 repression of specific genes were impacted. The alteration of these pathways in the SRC-2–/– uterus may have resulted in the developmental reprogramming of the uterus that culminated in the reduced response of the uterus to steroid hormone induction of the decidual reaction. Alternatively, because BMP and Wnt signaling have been implicated in regulating the decidual response (53), alteration of these gene pathways may, during induction, have impacted the decidual reaction. The results of our investigation provide significant insights into our understanding of the importance of SRCs in pregnancy and female reproduction.


   Acknowledgments
 
We gratefully acknowledge and thank Jun Qin for the antibody against SRC-2. We are grateful to Jinghua Li, Bryan Ngo, and Janet DeMayo, M.S., for technical assistance and Heather Franco for manuscript preparation. TIF2 knockout mice (referred to herein as SRC-2–/– for clarity) were constructed by the P. Chambon laboratory and given to us as a gift (31 33 ).


   Footnotes
 
This work was supported by the National Institutes of Health Grants U54HD007495 and PO1DK59820 (to F.J.D.) and National Institutes of Health Grant RO1-CA77530 and the Susan G. Komen Award BCTR0503763 (to J.P.L.).

Disclosure Statement: Authors have nothing to declare.

First Published Online June 7, 2007

Abbreviations: BMP, Bone morphogenetic protein; E2, estrogen; ER, estrogen receptor; P4, progesterone; PR, progesterone receptor; PRKO, PR knockout; SRC, steroid receptor coactivator protein; WIF1, Wnt inhibitory factor 1.

Received January 31, 2007.

Accepted for publication May 25, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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