Hoxa-10 Regulates Uterine Stromal Cell Responsiveness to Progesterone during Implantation and Decidualization in the Mouse

Hyunjung Lim, Liang Ma, Wen-ge Ma, Richard L. Maas and Sudhansu K. Dey

Department of Molecular and Integrative Physiology (H.L., W-G.M., S.K.D.) Ralph L. Smith Research Center University of Kansas Medical Center Kansas City, Kansas 66160-7338
Division of Genetics (L.M., R.L.M.) Brigham and Women’s Hospital and Harvard Medical School Boston, Massachusetts 02115


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hoxa-10 is an AbdominalB-like homeobox gene that is expressed in the developing genitourinary tract during embryogenesis and in the adult uterus during early pregnancy. Null mutation of Hoxa-10 in the mouse causes both male and female infertility. Defective implantation and decidualization resulting from the loss of maternal Hoxa-10 function in uterine stromal cells is the cause of female infertility. However, the mechanisms by which Hoxa-10 regulates these uterine events are unknown. We have identified two potential mechanisms for these uterine defects in Hoxa-10(-/-) mice. First, two PGE2 receptor subtypes, EP3 and EP4, are aberrantly expressed in the uterine stroma in Hoxa-10(-/-) mice, while expression of several other genes in the stroma (TIMP-2, MMP-2, ER, and PR) and epithelium (LIF, HB-EGF, Ar, and COX-1) are unaffected before implantation. Further, EP3 and EP4 are inappropriately regulated by progesterone (P4) in the absence of Hoxa-10, while PR, Hoxa-11 and c-myc, three other P4-responsive genes respond normally. These results suggest that Hoxa-10 specifically mediates P4 regulation of EP3 and EP4 in the uterine stroma. Second, since Hox genes are implicated in local cell proliferation, we also examined steroid-responsive uterine cell proliferation in Hoxa-10(-/-) mice. Stromal cell proliferation in mutant mice in response to P4 and 17ß-estradiol (E2) was significantly reduced, while epithelial cell proliferation was normal in response to E2. These results suggest that stromal cell responsiveness to P4 with respect to cell proliferation is impaired in Hoxa-10(-/-) mice, and that Hoxa-10 is involved in mediating stromal cell proliferation. Collectively, these results suggest that Hoxa-10 mutation causes specific stromal cell defects that can lead to implantation and decidualization defects apparently without perturbing epithelial cell functions.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hox genes are developmentally regulated transcription factors belonging to a multigene family. They share in common a highly conserved sequence element called the homeobox that encodes a 61-amino acid helix-turn-helix DNA-binding domain. While one Hox cluster (the HOM-C) is present in Drosophila, four mammalian Hox clusters (A, B, C, and D) exist on different chromosomes and have been generated by gene duplication (1). These genes follow a strict pattern of spatial and temporal colinearity during embryogenesis. For example, while genes at the 3'-end of each cluster are activated during early embryogenesis in the anterior region of the developing embryo, genes located toward the 5'-end are restricted to posterior regions of the embryo and are expressed during later stages of embryogenesis (1, 2). AbdominalB (AbdB) is the most 5'-gene within the Drosophila homeotic complex. In mammals, several AbdB-like genes exist at the 5'-ends of the Hox a, c, and d clusters corresponding to paralogous groups 9–13 (3). The AbdB genes constitute a distinct subfamily of homeobox genes that exhibit posterior domains of expression including the genital imaginal disc in Drosophila and the developing genitourinary system in vertebrates (4, 5).

Hoxa-10 is one AbdominalB-like homoeobox gene that is located in the Hoxa cluster and expressed in the developing genitourinary tract during mouse embryogenesis (3). Its distinct role in development has been defined by gene targeting experiments (6, 7). Hoxa-10-deficient mice exhibit male and female infertility along with a homeotic transformation of the lumbar vertebrae. Although the proximal uterus of Hoxa-10(-/-) mice shows partial homeosis into an oviduct-like structure, this is not the major cause of infertility in these mice. Hoxa-10 is strongly expressed in the stroma and decidua of the pregnant mouse uterus (6), and decidualization in Hoxa-10(-/-) mice is severely compromised during blastocyst implantation (8), thus reflecting a maternal requirement for Hoxa-10 in the periimplantation uterus. However, the mechanism by which Hoxa-10 regulates uterine stromal cell proliferation and differentiation during decidualization remains unknown. Recent investigations have revealed that Hoxa-10 (9, 10) and other AbdB-like Hoxa genes (9) are regulated by progesterone (P4) in the mouse and human endometrium. Hoxa-10 is induced in the mouse uterine stroma within 4 h of a P4 injection in a protein synthesis-independent fashion, and the up-regulation of Hoxa-10 by P4 is inhibited by the progesterone receptor (PR) antagonist RU-486, suggesting a requirement for PR for this induction (9). These studies imply that Hoxa-10 is a primary steroid hormone-responsive gene and that it is involved in implantation as a direct mediator of P4 actions. The adjacent AbdB gene, Hoxa-11, is also regulated by ovarian steroids in the uterine stroma (9), and Hoxa-11 mutant mice also exhibit female infertility originating from uterine defects similar to those in Hoxa-10(-/-) mice (11, 12).

A precise coordination between the establishment of uterine receptivity and blastocyst activation is essential to the process of implantation (13, 14). Ovarian P4 and estrogen play key roles in implantation and subsequent decidualization. While preovulatory estrogen secretion induces epithelial cell proliferation on day 1 of pregnancy, superimposition of estrogen on P4 priming on day 4 directs stromal cell proliferation and epithelial cell differentiation necessary for implantation in the mouse (15). This profile of uterine cell proliferation can be mimicked in the ovariectomized mouse uterus by ovarian steroids. For example, a single injection of 17ß-estradiol (E2) induces epithelial cell proliferation, while P4 induces stromal cell proliferation by 24 h, which is further potentiated by E2 (15). However, the mechanism by which these steroid hormones regulate uterine cell-specific proliferation and differentiation is unclear. The initial attachment reaction of the blastocyst trophectoderm with the luminal epithelium, which coincides with increased stromal vascular permeability and occurs around 2200–2300 h on day 4 of pregnancy, is followed by stromal cell proliferation and differentiation (decidualization) at the sites of blastocyst apposition (14). P4 is an absolute requirement for decidualization since PR-deficient mice fail to exhibit decidualization, while estrogen receptor-{alpha} (ER-{alpha}) deficient mice are capable of responding to a deciduogenic stimulus only in the presence of P4 (16 16A ).

Because of their vasoactive and mitogenic nature, PGs are implicated in implantation and decidualization (reviewed in Ref. 17). PGs are generated via cyclooxygenase (COX), which exists in two isoforms, COX-1 and COX-2 (18). COX-2, but not COX-1, is essential for implantation and decidualization (17). Among PGs, prostacyclin (PGI2) and PGE2 are believed to be important mediators of implantation (reviewed in Ref. 17). PGE2 binds and activates a set of functionally distinct cell surface receptors, EP1, EP2, EP3, and EP4, which are classified on the basis of their pharmacological responses to various agonists and antagonists of PGE2. They also exhibit different characteristics with respect to their structures, tissue distribution, and signal transduction mechanisms (19). While PGI2 can bind to one G protein-coupled receptor known as IP (19), PGI2 also functions as a ligand for peroxisome proliferator-activated receptors (PPAR{alpha} and PPAR{delta}), members of a nuclear hormone receptor superfamily (20, 21). Previous investigation on periimplantation defects in Hoxa-10(-/-) mice demonstrated poor vascular response and defective decidualization (8). These results suggested that altered PG signaling could be one cause of uterine failure in Hoxa-10(-/-) mice. Here we provide evidence that uterine stromal responsiveness to P4 with respect to both PG signaling and cell proliferation is defective in Hoxa-10(-/-) mice.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PGE2 Receptor Subtypes in the Uteri of Hoxa-10(-/-) Mice Are Aberrantly Expressed
We examined the expression of several implantation-related genes, such as LIF (22), HB-EGF (23), amphiregulin (Ar) (24), and COX-1 (25) in the Hoxa-10(-/-) mouse uterus on day 4 of pregnancy (Ref. 8 and data not shown). All of these genes are normally expressed in the uterine epithelium of Hoxa-10(-/-) mice. We have recently shown that PGI2 is the primary initiator of implantation and decidualization (17), and our recent investigation using COX-2 mice shows that PGE2 functions as an ancillary factor with PGI2 for implantation (H. Lim, R. A. Gupta, B. C. Paria, D. E. Moller, J. D. Morrow, R. N. DuBois, J. M. Trzaskos, and S. K. Dey, in preparation). Since IP is not detectable in the mouse uterus during implantation (H. Lim, R. A. Gupta, B. C. Paria, D. E. Moller, J. D. Morrow, R. N. DuBois, J. M. Trzaskos, and S. K. Dey, in preparation), we examined expression of PGE2 receptor subtypes in Hoxa-10(-/-) mice. Among the PGE2 receptor subtypes, EP2, EP3, and EP4 exhibit spatiotemporal expression in the periimplantation uterus, suggesting a role of PGE2 in implantation (26, 27). EP2 is solely expressed in the luminal epithelium primarily on days 4 and 5 of pregnancy (27). In contrast, EP3 is expressed in the mesometrial stroma and throughout the myometrium, while EP4 is expressed in the epithelium and stroma during the periimplantation period (26). In day 4 pregnant Hoxa-10(-/-) uteri, EP2 expression in the epithelium was normal (Fig. 1Go, a and b), but the exclusive mesometrial stromal localization of EP3 was lost with both mesometrial and lateral antimesometrial stromal expression at low levels (Fig. 1Go, c–f). Myometrial expression of this gene was also reduced in Hoxa-10(-/-) mice. With respect to EP4, the stromal expression was considerably down-regulated in Hoxa-10(-/-) uteri with little change in epithelial expression (Fig. 1Go, g–j).



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Figure 1. Expression of PGE2 Receptor Subtypes in Hoxa-10(-/-) Mice

In situ hybridization of EP2, EP3, and EP4 mRNAs in day 4 pregnant uteri of wild-type and Hoxa-10(-/-) mice is shown under darkfield at 20x (a–d, g, and h) or at 40x (e, f, i, and j). EP2 (a and b); EP3 (c–f); EP4 (g–j). Longitudinal sections (a–d, g, h); transverse sections (e,f, i, and j). Mesometrial pole of the uterus is directed toward the top of each picture. le, Luminal epithelium; s, stroma; myo, myometrium; cm, circular muscle; lm, longitudinal muscle. Similar results were observed in six mice.

 
These results could suggest that the aberrant EP3 and EP4 expression in the stroma reflects a global problem resulting from developmental alteration in uterine cell types. To address this, we examined the expression of several other genes, including tissue inhibitor of metalloproteinase-2 (TIMP-2), matrix metalloproteinase-2 (MMP-2), ER-{alpha}, and PR that are expressed in the stroma (16A, 28, 29). These genes were correctly expressed in the uteri of Hoxa-10(-/-) mice on day 4 of pregnancy (data not shown). Thus, the aberrant expression of EP3 and EP4 in the uterine stroma of Hoxa-10(-/-) mice appears to result specifically from stromal Hoxa-10 deficiency.

EP3 and EP4 Are Not Correctly Regulated by P4 in Ovariectomized Hoxa-10(-/-) Uteri
Since both EP3 and EP4 are regulated by P4 in the mouse uterus (26), we examined their regulation in Hoxa-10(-/-) uteri under steroid hormonal stimulation. Levels of ovarian P4 and estrogen and uterine responsiveness to these steroids are important for preparing the uterus and embryo for implantation (14). EP3 and EP4 expression in the uterus is up-regulated by P4 24 h after steroid injection of ovariectomized mice (26), and the uterine distribution of these genes after P4 injection resembles that on day 4 of pregnancy. In Hoxa-10(-/-) uteri, expression of EP3 in the stroma and myometrium by P4 was greatly reduced compared with that in wild-type mice (Fig. 2Go, a–d). In contrast, EP4 expression persisted at basal levels in ovariectomized wild-type uteri in the absence of steroids. However, P4 treatment up-regulated EP4 expression both in the stroma and epithelium in wild-type mice (Fig. 2Go, e and g). In Hoxa-10(-/-) uteri, EP4 expression was generally lower but especially in the stroma after P4 treatment (Fig. 2Go, f and h). No differences were noted in EP2 expression under similar conditions (data not shown). These results suggest that Hoxa-10 mediates the effects of P4 in regulating the correct expression of EP3 and EP4 in the uterine stroma. Moreover, the abnormal regulation of stromal EP3 and EP4 in Hoxa-10(-/-) uteri is apparently due neither to reduced levels of ovarian steroids nor aberrant expression of their nuclear receptors, since exogenous P4 injection in the ovariectomized Hoxa-10(-/-) mice did not correct this aberration (Fig. 2Go), and PR and ER-{alpha} are normally expressed in these mice (data not shown).



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Figure 2. Regulation of EP3 and EP4 mRNAs by Ovarian Steroid P4 in the Uteri of Ovariectomized Wild-Type and Hoxa-10(-/-) Mice

Wild-type and Hoxa-10(-/-) mice were injected with oil (vehicle, 100 µl) or P4 (2 mg/mouse) 2 weeks after ovariectomy. They were killed 24 h later, and uteri were collected for in situ hybridization. Darkfield photomicrographs are shown at 40x. EP3 (a–d, transverse sections); EP4 (e–h, longitudinal sections). Oil (a, b, e, and f); P4 (c, d, g, and h). le, Luminal epithelium; s, stroma; myo, myometrium; cm, circular muscle. Similar results were observed in three (EP3) or five (EP4) mice.

 
Altered P4 Responsiveness in the Stroma of Hoxa-10(-/-) Mice Is Restricted to Specific Genes
The above results suggest that intrinsic defects exist in Hoxa-10(-/-) uterine stroma in the regulation of uterine EP3 and EP4 expression. Since Hoxa-10 itself is regulated by P4 in the mouse and human endometrium (9, 10), it is possible that Hoxa-10 regulates the P4-induced expression of these genes. Alternatively, Hoxa-10(-/-) uteri could express a general defect in P4 responsiveness. Therefore, we analyzed the expression of three other uterine genes that are differentially regulated by P4 and/or E2 in the uterus. PR is induced in the ovariectomized mouse uterus in a cell-specific manner within 6 h of P4 and/or E2 administration (Ref. 29 and our unpublished results). Hoxa-11, another AbdB-like homeodomain gene, is also up-regulated by P4 and/or E2 by 6 h (9). c-myc is induced by P4 and E2 by 6 h in the uterus (30). Expression of PR (2 h, 6 h, and 24 h), Hoxa-11 (6 h) and c-myc (6 h) was examined by ribonuclease (RNase) protection assay and/or by in situ hybridization in uteri of ovariectomized wild-type and Hoxa-10(-/-) mice treated with P4 with or without E2. PR was induced at similar levels in the ovariectomized wild-type and Hoxa-10(-/-) uteri treated with P4 and E2, reaching a peak at 6 h (Fig. 3AGo). Further, in situ hybridization revealed similar localization and levels of Hoxa-11 in P4-treated wild-type and Hoxa-10(-/-) uteri (Fig. 3BGo). Lastly, the up-regulation of c-myc by P4 and E2 at 6 h also remained indistinguishable between wild-type and Hoxa-10(-/-) mice (Fig. 3CGo). These results further confirm that the aberrant regulation of EP3 and EP4 in Hoxa-10(-/-) uteri is a specific consequence of stromal deficiency of Hoxa-10 and that Hoxa-10 mediates the steroid hormonal regulation of these genes in the uterine stroma.



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Figure 3. Uterine Responsiveness to P4 in Hoxa-10(-/-) Mice

A, Quantitation of the steroidal regulation of PR in the uteri of ovariectomized wild-type and Hoxa-10(-/-) mice by RNase protection assay. Mice were injected with oil or P4 (2 mg/mouse) plus E2 (100 ng/mouse) 2 weeks after ovariectomy. They were killed at indicated times, and uteri were collected for RNA extraction. Three sets of RNase protection assays were analyzed, and band intensities were quantitated by phosphorimager and normalized for loading differences with rpL19. Relative values after normalization are shown (mean ± SEM). B, Regulation of Hoxa-11 by P4 in the uteri of ovariectomized wild-type and Hoxa-10(-/-) mice. Ovariectomized mice were injected with oil or P4 (2 mg/mouse). They were killed 6 h later, and uteri were collected for in situ hybridization. Darkfield photomicrographs are shown at 40x. le, Luminal epithelium; s, stroma; myo, myometrium. Similar results were observed in three mice in each group. C, Quantitation of the steroidal regulation of c-myc in the uteri of ovariectomized wild-type and Hoxa-10(-/-) mice by RNase protection assay. Mice were injected with oil or P4 (2 mg/mouse) plus E2 (100 ng/mouse) 2 weeks after ovariectomy. They were killed 6 h later, and uteri were collected for RNA extraction. Three sets of RNase protection assays were analyzed, and band intensities were quantitated by phosphorimager and normalized for loading differences with rpL19. Relative values after normalization are shown (mean ± SEM).

 
Stromal Cell Proliferation in Response to P4 and E2 is Reduced in Uteri of Ovariectomized Hoxa-10(-/-) Mice
Epithelial cell proliferation in the ovariectomized mouse uterus is induced by E2, while that of stromal cells is induced by P4 and further potentiated by E2; these profiles closely mimic the patterns observed during early pregnancy (15). We compared cell proliferation profiles in response to P4 and/or E2 between ovariectomized wild-type and Hoxa-10(-/-) uteri. While an E2 injection induced epithelial cell proliferation at equal levels in both wild-type and Hoxa-10(-/-) uteri, stromal cell proliferation in response to P4 and E2 was {approx}7-fold lower in Hoxa-10(-/-) mice (Fig. 4Go and Table 1Go). These data indicate that Hoxa-10 is required for basal and for P4-induced stromal cell proliferation.



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Figure 4. Uterine Cell Proliferation in Wild-Type and Hoxa-10(-/-) Mice after Steroid Treatments

Mice were injected with oil, E2 (100 ng/mouse), or P4 (2 mg/mouse) plus E2 2 weeks after ovariectomy. Twenty-two hours after steroid injection, they received an intraperitoneal injection of [methyl-3H]thymidine (25 µCi/0.1 ml saline) and were killed 2 h later. Nuclear uptake of [3H]thymidine was detected in uterine sections by autoradiography after 7 days of exposure. le, Luminal epithelium; s, stroma; myo, myometrium. Similar results were observed in six mice (see Table 1Go).

 

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Table 1. Quantitation of Nuclear [3H]Thymidine Uptake in Steroid Hormone-Treated Wild-Type and Hoxa-10(-/-) Mice

 
COX-2 Expression Is Aberrant in Hoxa-10(-/-) Uteri at the Initial Stage of Decidualization
Since PGs are implicated in cell proliferation (31, 32), the involvement of Hoxa-10 in cell proliferation could be via a PG-signaling pathway. PGs produced via uterine COX-2 are critical for implantation and decidualization (17) and are also implicated in the proliferation and transformation of several cell types (reviewed in Refs. 33, 34). Thus, we examined COX-2 induction in Hoxa-10(-/-) mice using an experimentally induced decidualization model. A biphasic induction of COX-2 was noted in the day 4 pseudopregnant mouse uterus after intraluminal oil infusion as a deciduogenic stimulus. COX-2 expression was induced in the epithelium of the oil-infused horn at 2 h, but disappeared by 8 h (17). A second phase of COX-2 expression was focally induced in the subepithelial stroma at 24 h (Fig. 5BGo), resembling COX-2 induction at the time of blastocyst attachment reaction (25). In Hoxa-10(-/-) mice, epithelial COX-2 induction at 2 h was normal, but stromal COX-2 induction at 24 h was considerably reduced or absent in Hoxa-10(-/-) mice (Fig. 5Go, A and B). A significant reduction of stromal COX-2 was also confirmed by RNase protection assay (Fig. 5CGo). This provides evidence that loss of COX-2 in the absence of Hoxa-10 is related to defective decidualization in these mice. Alternatively, this could be a consequence of decidualization failure in Hoxa-10(-/-) mice.



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Figure 5. Induction of COX-2 mRNA in Day 4 Pseudopregnant Wild-Type And Hoxa-10(-/-) Uteri with or without Intraluminal Oil Infusion

A, Northern blot hybridization of COX-2 mRNA at 2 h after oil infusion. Pseudopregnant wild-type or Hoxa-10(-/-) mice received an intraluminal oil infusion on the morning of day 4. Two hours later, infused and noninfused uteri were collected separately and pooled from eight to nine mice. They were subjected to RNA extraction and Northern blot hybridization. Lane 1, Wild-type noninfused horns; lane 2, wild-type infused horns; lane 3, Hoxa-10(-/-) noninfused horn; lane 4, Hoxa-10(-/-) infused horns. rpL7 serves as a loading control. B, In situ hybridization of COX-2 mRNA in the stroma at 24 h after intraluminal oil infusion. Pseudopregnant wild-type or Hoxa-10(-/-) mice received an intraluminal oil infusion on the morning of day 4. Uteri were collected 24 h later for in situ hybridization. le, Luminal epithelium; s, stroma; myo, myometrium. Reduced stromal induction of COX-2 was noted in 10 of 12 mice. C, RNase protection assay of stromal COX-2 induction in wild-type and Hoxa-10(-/-) mice. Pseudopregnant wild-type or Hoxa-10(-/-) mice received an intraluminal oil infusion on the morning of day 4. Uteri were collected 24 h later for RNase protection assays. Three sets of assays were analyzed, and band intensities were quantitated by phosphorimager and normalized for loading differences with rpL19. Relative values after normalization are shown (mean ± SEM).

 
Supplementation of PGs Cannot Restore Decidualization in Hoxa-10(-/-) Mice
Because stromal COX-2 was poorly induced in the Hoxa-10(-/-) uteri, we administered PGE2 and/or carbaprostacyclin (cPGI, a stable agonist of PGI2) to Hoxa-10(-/-) mice in an attempt to correct the decidualization defects that were observed in the experimentally induced decidualization model. PGE2 and/or cPGI was administered from days 4–7 of pseudopregnancy in Hoxa-10(-/-) mice after an intraluminal oil infusion on day 4. Supplementation of PGE2 and/or cPGI was ineffective in improving the decidual response in Hoxa-10(-/-) mice. While four of five wild-type mice showed {approx}13 fold uterine weight increases in response to intraluminal oil infusion, none of the five Hoxa-10(-/-) mice showed any response. Further, cPGI (n = 4), PGE2 (n = 5), or cPGI plus PGE2 (n = 5) treatment did not improve the decidual response in these mutants. The results are consistent with the idea that the defective decidualization in Hoxa-10(-/-) mice reflects altered downstream signaling of PGs due to aberrant expression of their receptors.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Across metazoa, functions of Hox homeoproteins are conserved as factors that specify embryonic segmental structures (1). Genetic studies have provided insight into the mechanisms by which these transcription factors affect such precisely regulated cellular events. However, apparent differences exist in the way Drosophila Hox genes exert their actions during development from those of mammalian Hox genes (35). One recent view on mammalian Hox functions envisages their roles as local regulators of cell proliferation (35), although their definitive functions in different systems remain unknown. Defective implantation and decidualization resulting in female infertility in the absence of Hoxa-10 (8) demonstrate an unexpected function for a Hox gene.

Appropriate levels of P4 and responsiveness of the uterus to this steroid are absolute requirements for decidualization (16 16A, 17). The failure of P4 to restore decidualization in Hoxa-10(-/-) mice (8) and the regulation of uterine Hoxa-10 by P4 (9) suggest that this gene is involved in mediating some important P4 actions in the uterus, and the identification of functional PR response elements (PREs) in the Hoxa-10 and Hoxa-11 intergenic region supports this notion (L. Ma and R. L. Maas, unpublished). Since Hoxa-10 is expressed in stromal cells in the periimplantation uterus and after steroid hormonal stimulation (6, 9), aberrant expression of EP3 and EP4 under these conditions in Hoxa-10(-/-) mice suggests that this homeobox protein acts as a direct or indirect mediator of P4 function in regulating these genes during implantation and decidualization. This is consistent with the observation that P4 induction of Hoxa-10 temporally precedes that of EP3/EP4. The correct expression of other P4-regulated uterine genes expressed in the epithelium (EP2 and Ar) or in the stroma (PR, Hoxa-11, and c-myc) in Hoxa-10(-/-) uteri further supports this hypothesis. The normal expression of ER and PR indicates that aberrant expression of EP3 and EP4 in the stroma is not the result of altered expression of these nuclear steroid receptors.

Reduced stromal cell proliferation in response to P4 and E2 in Hoxa-10(-/-) mice could potentially represent a consequence of altered PG signaling and may contribute to the defective decidual response in these mice. Alternatively, defective stromal cell proliferation in these mice could constitute a distinct phenomenon unrelated to PG signaling, since mammalian Hox genes are implicated in cell proliferation events (35). Cyclin D3, one of the G1 phase cyclins, is expressed in stromal cells at the onset of decidualization and is likely to activate cell cycle progression during this time (36). We have recently observed that expression of cyclin D3 fails to be up-regulated in Hoxa-10(-/-) uteri after application of a deciduogenic stimulus (36). This is also consistent with impaired stromal cell proliferation and points toward an underlying basis for the defective decidual response in Hoxa-10(-/-) mice.

Uterine COX-2 is induced by activated blastocysts at the time of the attachment reaction and produces PGs that are essential for implantation and decidualization (17, 25). Uterine and/or embryonic COX-2 also appears to be important for implantation in various species including sheep, mink, skunk, and baboon (37, 38, 39, 40). COX-2 exhibits a unique biphasic cell-specific induction at 2 h and 24 h during the initial stages of decidualization (Ref. 17 and Fig. 5Go), and the stromal cell expression at 24 h resembles the expression during blastocyst implantation (17, 25). The loss of stromal COX-2 expression in Hoxa-10(-/-) mice is intriguing, since these COX-2 expressing cells exhibit the first decidual cell reaction (17). Since uterine COX-2 is not directly regulated by P4 and/or E2 (25), loss of stromal COX-2 in these mice could be an indirect consequence of defective decidualization. Alternatively, the loss of stromal COX-2 in Hoxa-10(-/-) mice could indicate that Hoxa-10 regulates COX-2 in this cell type. The second speculation is supported by the observation that Hoxa-10 gene is correctly expressed in the COX-2(-/-) uteri (17), implying that Hoxa-10 is functionally upstream of COX-2 expression in the subepithelial stroma.

The inability of exogenously administered PGs to improve decidual responsiveness in Hoxa-10 mutants could be due to inappropriate selection of time and doses, rapid metabolism, and/or suboptimal delivery of these agents to the target cells. On the other hand, the defective decidualization in Hoxa-10(-/-) mice could result from altered PGE2 signal transduction due to aberrant EP receptor expression. This phenotype of Hoxa-10deficiency is clearly distinct from that of COX-2(-/-) mice (17). COX-2(-/-) mice, which exhibit normal uterine cell proliferation and expression of EP3 and EP4, respond to exogenous cPGI and PGE2 with improved implantation and decidualization (Ref. 17 and H. Lim, R. A. Gupta, B. C. Paria, D. E. Moller, J. D. Morrow, R. N. DuBois, J. M. Trzaskos, and S. K. Dey, in preparation). Therefore, aberrant expression of EP3 and EP4 in Hoxa-10(-/-) uteri suggests that signaling via these two receptors is important for decidualization. Although EP3-deficient female mice exhibit apparently normal reproductive performance (41), EP4-deficient mice exhibit neonatal lethality and are therefore uninformative for this function (42). Thus, EP4 could represent a potential candidate for PGE2 signaling in decidualization. Although the PGI2 cell surface receptor IP and nuclear receptor PPAR{alpha} are not detected in the mouse uterus at the time of implantation, another PGI2 nuclear receptor PPAR{delta} is expressed in stromal cells around the blastocyst with the initiation of the attachment reaction and subsequent decidualization. We have also found that COX-2 and PPAR{delta} are coordinately expressed in stromal cells after application of a deciduogenic stimulus (H. Lim, R. A. Gupta, B. C. Paria, D. E. Moller, J. D. Morrow, R. N. DuBois, J. M. Trzaskos, and S. K. Dey, in preparation). These observations suggest that PGI2 signaling in implantation and decidualization is mediated via PPAR{delta}, but not by IP or PPAR{alpha}. We have preliminary evidence that PPAR{delta}, like COX-2, fails to be up-regulated in stromal cells after application of a deciduogenic stimulus in Hoxa-10(-/-) mice (data not shown). Thus, aberrant expression of EP3, EP4, PPAR{delta}, and COX-2 in Hoxa-10(-/-) uteri suggests that PG signaling plays an important role in implantation and decidualization and that Hoxa-10 is involved in regulating these signaling systems. Finally, implantation and decidualization defects in Hoxa-10(-/-) mice are also correlated with defective stromal cell proliferation in response to steroid hormones. Based on our observation of reduced expression of cyclin D3 with the onset of decidualization in Hoxa-10(-/-) mice (36), this result is consistent with a defect in progression through the G1 phase of the cell cycle. Although PGs are involved in cell proliferation, whether defective cell proliferation in Hoxa-10(-/-) mice results from defective PG signaling will require further investigation.

The process of implantation involves regulated mitogenesis and vascular permeability changes in the uterus, and ovarian steroids play pivotal roles in these uterine events (14). P4 mediates a variety of female reproductive functions as demonstrated in PR-deficient mice (16). Our results show that Hoxa-10(-/-) mice provide a good model to define the role of P4 actions in the uterine stroma during implantation. Interestingly, although a smaller percentage of Hoxa-10(-/-) mice achieve successful pregnancy, about 40% of Hoxa-10(-/-) mice succeed in initiating the implantation reaction (8), suggesting that stromal defects in Hoxa-10(-/-) mice do not completely negate the functions of luminal epithelial cells for initial blastocyst contact. It is possible that Hoxa-10 induces genes that are important for stromal cell proliferation and differentiation in a P4-dominant environment. Our results potentially identify the PG-signaling system as acting functionally downstream of uterine Hoxa-10 in implantation. Whether EP3, EP4, PPAR{delta}, or COX-2 are directly or indirectly regulated by Hoxa-10 requires further investigation. Hoxa-10 is also implicated in the proliferation and differentiation of the myeloid lineage during hematopoiesis (43, 44), implying that this protein may also be capable of functioning similarly in both contexts. Since Hox functions during embryogenesis have been considered as regulators of local cell proliferation (35), the uterine cell proliferative defect in Hoxa-10(-/-) mice could provide a potentially powerful system with which to study the role of Hox genes in cell proliferation and cell cycle control. Collectively, our present findings suggest a novel role of Hoxa-10 in mediating certain actions of P4 in the uterine stroma with respect to implantation and decidualization.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Gene-Targeted Mice
The disruption of the Hoxa-10 gene was performed by insertion of a neomycin resistance cassette into an XhoI site within the homeobox by homologous recombination in 129/SvJ ES cells and generation of chimeric mice (6). PCR analysis of tail genomic DNA determined the genotypes. All of the mice used were housed in the animal care facility at the University of Kansas Medical Center according to NIH and institutional guidelines on the care and use of laboratory animals. Females were mated with fertile or vasectomized males of the same strain to induce pregnancy or pseudopregnancy (day 1 = vaginal plug), respectively.

Hybridization Probes
For Northern hybridization and RNase protection assay, 32P-labeled antisense cRNA probes were generated, while for in situ hybridization, sense and antisense 35S-labeled cRNA probes were generated using the appropriate polymerases. Mouse-specific cDNAs to c-myc, LIF, Ar, Hoxa-10, Hoxa-11, COX-1, COX-2, PGE2 receptor subtypes (EP2, EP3, EP4), estrogen receptor-{alpha} (ER-{alpha}), progesterone receptor (PR), tissue inhibitor of metalloproteinase-2 (TIMP-2), matrix metalloproteinase-2 (MMP-2), rpL7, and rpL19 were used as templates for generating probes for Northern blot hybridization, in situ hybridization, or RNase protection assay (9, 17, 24, 25, 26, 27, 28, 29, 30).

In Situ Hybridization
In situ hybridization was performed as described previously (23). Briefly, uteri were cut into 4–6 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. The 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 {approx}2 x 109 dpm/µg). After hybridization and washing, the sections were incubated with RNase A (20 µg/ml) at 37 C for 20 min. RNase A-resistant hybrids were detected by autoradiography using Kodak NTB-2 liquid emulsion (Eastman Kodak Co., Rochester, NY). Sections hybridized with the corresponding sense probe served as negative controls. Slides were poststained with hematoxylin and eosin.

Northern Blot Hybridization
Total RNA was extracted from whole uteri pooled from 10–15 mice at indicated times by a modified guanidine thiocyanate procedure (23, 45). Total RNA (6 µg) was denatured, separated by formaldehyde-agarose gel electrophoresis, transferred to nylon membranes, and cross-linked by UV irradiation (Spectrolinker, XL-1500, Spectronics Corp., Westbury, NY). The blots were prehybridized, hybridized with 32P-labeled antisense cRNA probe (specific activities {approx}2 x 109 dpm/µg), and washed as described previously (23). After hybridization, the blots were washed under stringent conditions, and the hybrids were detected by autoradiography. The blots were stripped and rehybridized with rpL7 probe as described previously (27).

RNase Protection Assay
32P-labeled cRNA probes were prepared as described above. Analyses were performed as described previously (9, 46). For each lane, 20 µg total RNA were hybridized for 16 h at 45 C simultaneously with 3 x 105 cpm of RNA probes for each of c-myc, PR, or COX-2 and with 2 x 104 cpm of rpL19 RNA probe and then digested with 20 µg/ml RNase A and 1.5 µg/ml RNase T1. Protected fragments were electrophoresed in 6% denaturing polyacrylamide gel and analyzed by autoradiography. Band intensities were quantitated by phosphorimager and normalized for loading differences with rpL19.

Expression of Uterine Genes on Day 4 of Pregnancy in Hoxa-10(-/-) Mice
Sections of day 4 (0900 h) pregnant uteri from wild-type or Hoxa-10(-/-) mice were processed for in situ hybridization for LIF, Ar, COX-1, EP2, EP3, EP4, TIM-2, MMP-2, ER-{alpha}, and PR mRNAs.

Uterine Responsiveness to E2 and P4 in Hoxa-10(-/-) Mice
To determine whether Hoxa-10(-/-) mice respond appropriately to P4, wild-type and Hoxa-10(-/-) mice were ovariectomized and treated with sesame oil (vehicle) or P4 (2 mg/mouse) with or without E2 (100 ng/mouse) after 2 weeks of rest. Sesame oil and steroid hormones were purchased from Sigma Chemical Co. (St. Louis, MO). Uteri were collected 2, 6, or 24 h after the last injection. Induction of PR (2, 6, and 24 h), Hoxa-11 (6 h), c-myc (6 h) EP3 (24 h), and EP4 (24 h) genes was assayed by RNase protection and/or in situ hybridization.

To examine uterine cell-specific proliferation in response to E2 and/or P4, ovariectomized wild-type or Hoxa-10(-/-) mice were given an injection of E2 or P4 plus E2. After 22 h, they received an injection of [methyl-3H]thymidine (25 µCi/0.1 ml saline ip, specific activity, 40 mCi/mmol; RPI Corp., Mount Prospect, IL) and were killed 2 h later. Uteri were flash frozen and fixed in 4% paraformaldehyde after sectioning. Nuclear uptake of [3H]thymidine was detected in uterine sections by autoradiography (15) after 7–10 days of exposure. The autoradiographic signals (silver grains) were quantitated under a darkfield using the OPTIMA II program with an image analysis system (47).

Induction of COX-2 in the Hoxa-10(-/-) Uteri after Application of a Deciduogenic Stimulus
To examine COX-2 induction in the Hoxa-10(-/-) uteri in response to a deciduogenic stimulus, wild-type and Hoxa-10(-/-) mice received intraluminal oil (25 µl) on day 4 of pseudopregnancy. Uteri were collected at 2 or 24 h after the oil infusion for Northern blot hybridization, in situ hybridization, or RNase protection assay.

Decidual Response in Hoxa-10(-/-) Mice after Supplementation of PGs
To induce decidualization, sesame oil (25 µl) was infused intraluminally in one uterine horn on day 4 of pseudopregnancy; the contralateral horn served as control. Mice were killed on day 8, and uterine weights of the infused and noninfused (control) horns were recorded to assess the extent of decidualization. PGE2 and/or cPGI (Cayman Chemical Co., Ann Arbor, MI) were prepared in 10% EtOH-90% saline solution (20 µg/injection) and supplemented intravenously at 1700 h on day 4 of pseudopregnancy followed by an intraperitoneal injection on days 5–7.


    FOOTNOTES
 
Address requests for reprints to: S. K. Dey, Ph.D., Department of Molecular and Integrative Physiology, MRRC 37/3017, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160-7338. E-mail: sdey{at}kumc.edu

This work was supported by NICHD/NIH grants as part of the National Cooperative Program on Markers of Uterine Receptivity for Blastocyst Implantation [(HD-29968) and HD-12304 to S.K.D.], and by NICHD Grant HD-35580 (to R.L.M.). H. L. was supported by a Kansas Health Foundation predoctoral fellowship, and L. M. is supported by an NIH National Research Service Award (1F32 HD-08264–01). Center grants in Reproductive Biology (HD-33994) and Mental Retardation (HD-02528) at the University of Kansas Medical Center provided access to various core facilities.

Received for publication January 6, 1999. Revision received February 23, 1999. Accepted for publication February 25, 1999.


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