Regional development of uterine decidualization: molecular signaling by Hoxa-10.

The establishment of a differentiated uterus for supporting embryo development and implantation is primarily dependent on the coordinated effects of ovarian estrogen and progesterone (P 4 ) ( Psychoyos, 1973 ; Paria et al., 1993 ). In the rodent, the first conspicuous sign that the implantation process has been initiated is an increased endometrial vascular permeability at the sites of blastocyst implantation. This can be visualized as discrete blue bands along the uterus after an intravenous injection of a blue dye solution ( Psychoyos, 1973 ; Huet and Dey, 1987 ; Paria et al., 1993 ). This increased localized vascular permeability coincides with the initial attachment reaction between the uterine luminal epithelium and blastocyst trophectoderm ( Enders and Schlafke, 1967 ) and is considered to be one of the earliest prerequisite events in the implantation process ( Psychoyos, 1973 ). In the mouse, the attachment reaction occurs in the evening (2200–2300 hr) of day 4 of pregnancy, and is preceded by uterine luminal closure which in turn results in an intimate apposition of the trophectoderm with the luminal epithelium ( Enders and Schlafke, 1967 ; Finn and McLaren, 1967 ; Psychoyos, 1973 ; Enders, 1976 ; Das et al., 1994 ). The attachment reaction is followed by localized stromal cell decidualization and luminal epithelial apoptosis at the sites of blastocyst implantation ( Parr et al., 1987 ). This results in subsequent adherence and penetration by trophoblast cells through the underlying basement membrane ( Enders and Schlafke, 1967 ). Invasion of trophoblast cells continues through the stroma in a regulated manner by the remodeling of the extracellular matrix. In mice, the molecular and cellular events leading to the uterine receptivity for implantation depend upon the priming of the uterus by P 4 and estrogen [estradiol-17β, (E 2 )] ( Dey et al., 2004 ). A similar steroid hormonal regulation is required for the cell-type specific proliferation and differentiation in the uterus during the periimplantation period ( Finn and Martin, 1967 ; Huet-Hudson et al., 1989 ). For example, preovulatory ovarian E 2 secretion causes proliferation and differentiation of the epithelium during the first 2 days of pregnancy. In contrast, rising P 4 levels from the newly formed corpora lutea result in a shift of proliferation from the epithelium to the stroma on Day 3. Stromal cell proliferation is potentiated by preimplantation ovarian E 2 on day 4 (the day of implantation). This E 2 secretion is also necessary for the increased endometrial capillary permeability at the sites of implanting blastocysts, and decidualization in the presence of the blastocyst or other deciduogenic stimuli ( Finn and Martin, 1967 ; Yoshinaga, 1980 ; Huet-Hudson et al., 1989 ). In order to initiate the implantation, the onset of the uterine receptive state occurs only for a limited period during pregnancy, pseudopregnancy, or when the uterus is appropriately prepared with P 4 and E 2 . For example, in mice, uterine receptivity can be divided into neutral, receptive, and non-receptive (refractory) phases ( Dey et al., 2004 ). Normally, uteri are prereceptive on day 3 of pregnancy or pseudopregnancy under the rising ovarian P 4 levels. On Day 4, the P 4 -primed uterus becomes receptive when complemented with preimplantation ovarian E 2 secretion. Thus, E 2 is a critical determinant to specify the window of uterine receptivity for implantation ( Ma et al., 2003 ). In fact, studies have shown that estrogen at different concentrations can initiate implantation; however, the duration of the window of uterine receptivity can be prolonged at lower concentrations of estrogen but rapidly closes at its higher levels ( Ma et al., 2003 ). In contrast, P 4 alone is adequate for the induction of implantation in the hamster, guinea pig, rabbit, and pig ( Deanesly, 1960 ; Orsini and Meyer, 1962 ; Perry et al., 1973 ; George and Wilson, 1978 ). E 2 of embryonic origin may participate in implantation in these species ( Pakrasi et al., 1985 ; Reese et al., 2008 ). In this regard, it is important to note that there is no evidence for E 2 synthesis in the mouse blastocyst ( Stromstedt et al., 1996 ). Blastocysts implant only in the receptive uterus (Day 4), which then automatically enters into the nonreceptive state on Day 5. Similar uterine phases can be produced by ovariectomy on the morning of day 4 of pregnancy or pseudopregnancy before the preimplantation E 2 secretion. Under this condition, blastocysts in the pregnant uterus or transferred blastocysts into the pseudopregnant uterus undergo dormancy and fail to initiate the attachment reaction (delayed implantation). This condition can be maintained by continued P 4 treatment when the uterus remains in the neutral (similar to prereceptive) state, but is terminated by a single E 2 injection with blastocyst activation and attainment of uterine receptivity. Current molecular and genetic evidence indicates that several developmentally important, and E 2 /P 4 regulated-signaling molecules, including homeobox transcription factors, cytokines, growth factors, lipid mediators serve as autocrine, paracrine, and juxtacrine factors, help to specify uterine receptivity ( Lim et al., 2002 ; Paria et al., 2002 ; Dey et al., 2004 ; Wang and Dey, 2006 ; Kennedy et al., 2007 ; Lee et al., 2007 ).

Embryo implantation is a complex process that requires two-way interactions between the implantation-competent blastocyst and the receptive uterus ( Psychoyos, 1973 ; Dey, 1996 ), and involves complex growth regulatory mechanisms leading to molecular, cellular, physiological, and functional changes in the uterus and embryo. Any aberration of this process leads to the human infertility, which remains a major global concern both socially and economically. While the underlying cause of human infertility has been potentially challenged by successful utilization of in vitro fertilization and embryo-transfer techniques; the success rate of implantation remains disappointingly low, which is considered primarily due to our poor understanding regarding the uterine receptivity during the transfer of embryos. Therefore, there is a clear need to unravel the complexities of basic mechanisms of embryo development and implantation. Although a significant progress has been made during the last two decades ( Paria et al., 2002 ; Dey et al., 2004 ; Wang and Dey, 2006 ), a deeper understanding in needed to optimize success.

Endometriosis is a common gynecological disease that remains an enigma. The pathogenesis of this disease is characterized by endometrial tissue growth outside the uterus and results in inflamed lesions, pain, and reduced fertility. Although heightened estrogenic activity and/or reduced P 4 responsiveness are considered to be involved in the etiology of this disease, neither the extent of their participation nor the underlying mechanisms are clearly understood. Previous studies have shown that the uteri form endometriotic patients failed to show the mid-secretory rise of Hoxa-10 expression as compared to the controls ( Taylor et al., 1999 ), indicating that Hoxa-10 may contribute to the etiology of infertility in patients with endometriosis. In this regard, recent studies suggest that epigenetic alteration by increased DNA methylation in the CpG clusters of Hoxa-10 gene promoter may be responsible for this aberration of Hoxa-10 expression ( Wu et al., 2005 ; Kim et al., 2007 ). Utilizing a proteomic approach, we have recently identified the immunophilin FKBP52 as one of the Hoxa-10-mediated signaling molecules in the mouse uterus ( Daikoku et al., 2005 ). Genetic deletion of FKBP52 in mice establishes its critical role in embryo implantation, and they exhibit an increased P 4 resistance in uterine functions ( Tranguch et al., 2005 ). Using FKBP52 −/− mice, recent studies showed that the loss of FKBP52 enhances the growth of endometriotic lesions with increased inflammation, cell proliferation, and angiogenesis ( Hirota et al., 2008 ). Additionally, studies also showed a remarkable downregulation of FKBP52 in cases of human endometriosis, suggesting a potential role of FKBP52 in the etiology of human endometriosis. Furthermore, these studies provide evidence that P 4 resistance is favorably implicated in endometriosis and other gynecological diseases. Overall, these results suggest that Hoxa-10 may be a potential therapeutic target to alleviate the problem with endometriosis. Recent studies by Penna et al. (2009) have shown that Calpain5, a member of the cytoplasmic cysteine protease family involves in cellular differentiation and apoptosis ( Suzuki et al., 2004 ), is expressed in the human endometrial stroma during the menstrual cycle and in the decidua. Based on transfection experiments in human endometrial stromal cells for the expression or suppression of HOXA 10, it has been revealed that Calpain5 acts as a downstream target of HOXA 10. Furthermore, studies have shown that the expression of Calpain5, like that of HOXA 10, is aberrantly downregulated in human endometriotic tissues ( Gui et al., 1999 ; Taylor et al., 1999 ; Kim et al., 2007 ; Penna et al., 2009), suggesting that a HOXA 10–Calpain5 connection may be either directly or indirectly involved in the progression of endometriosis.

The presence of blastocysts or artificial stimuli can trigger differentiation of stromal cells to decidual cells in the receptive uterus. The decidual cell reaction or decidualization is always preceded by increased uterine vascular permeability ( Psychoyos, 1973 ; Kennedy, 1986 ). Pulse-labeling experiments with 3 H-thymidine suggest that decidual cells originate from undifferentiated stromal cells ( Galassi, 1968 ). The decidualization is first initiated at the antimesometrial site where blastocysts implant, which then spreads to the mesometrial pole, the presumptive site of placentation. Normally, this defines the implantation chamber in an antimesometrial–mesometrial direction in alignment with the anterior–posterior axis of the embryo. Generally, the decidualization process is characterized by stromal cell proliferation and differentiation into specialized type of cells (decidual cells) with polyploidy. In the mouse, the differentiating stromal cells in the antimesometrial location at the site of blastocyst form the primary decidual zone (PDZ) in the afternoon on Day 5. PDZ is avascular and epithelioid in nature ( Paria et al., 1999 ). By Day 6, stromal cells next to the PDZ continue to proliferate and differentiate into polyploid decidual cells, forming a zone around the PDZ, termed the secondary decidual zone (SDZ). SDZ is fully developed by Day 7, while the PDZ degenerates progressively by apoptosis and by day 8 most of these cells disappear. After Day 8, the placental and embryonic growth slowly replaces the SDZ. A similar, although not identical ( Paria et al., 2001 ; Bany and Cross, 2006 ), pattern of decidualization can be initiated by the application of artificial stimuli to a receptive pseudopregnant uterus, or one that has been appropriately primed by ovarian steroids ( Dey, 1996 ). Using these model systems, cellular and molecular changes that occur during decidualization under the control of P 4 and E 2 can be examined. Numerous signaling molecules including homeo-box transcription factors, cell-cycle molecules, and growth factors have been proposed to play roles in the progression of this process ( Lim et al., 2002 ; Paria et al., 2002 ; Dey et al., 2004 ; Wang and Dey, 2006 ).

Based on above discussions, a working model for regional developmental mechanism of decidualization by Hoxa-10 is presented in Figure 5 . Hoxa-10 is expressed under the direction of ovarian P 4 in uterine stromal cells either prior to or during decidualization. Hoxa-10 mediates implantation-induced signaling pathways that include the regulation of stromal cell proliferation/differentiation and uNK cell differentiation, either in parallel or in a dependent fashion, to ultimately control a full complement of regional decidual development.

The role of maternal nutrition to establish pregnancy and maintenance of embryonic development has been well documented, yet very few reports show how specific nutrients can affect early implantation and decidualization. In this regard, the role of vitamin D in the reproductive function and pregnancy establishment in rodents is interesting ( Halloran and DeLuca, 1980 ; Yoshizawa et al., 1997 ; Daftary and Taylor, 2006 ). While studies have implicated that vitamin D is not absolutely necessary for reproduction, female rats fed a vitamin D-deficient diet show diminished reproduction success with reduced litter size ( Halloran and DeLuca, 1980 ; Daftary and Taylor, 2006 ). Additionally, female knockout mice for vitamin D receptors are unable to reproduce and exhibit defects of uterine development and decidualization ( Yoshizawa et al., 1997 ). Similarly, the null mice for 25-hydroxyvitamin D-1a-hydrolase, an enzyme necessary for the synthesis of vitamin D from its precursor 25-hydroxyvitamin D, show uterine hypoplasia and infertility primarily due to combined uterine and ovarian defects ( Panda et al., 2001 ). Moreover, studies have shown that intraluminal injection of vitamin D in the rat uterus on Day 5 of pseudopregnancy enhances uterine decidual response ( Kwiecinksi et al., 1989 ; Halhali et al., 1991 ). Overall, these data suggest that vitamin D has an essential role in fertility, necessary for differentiation of decidual cells. Recent studies have implicated a direct role of vitamin D in the regulation of HOXA-10 in human endometrial stromal cells ( Du et al., 2005 ). Studies have shown that the exposure of vitamin D to the human endometrial stromal cells causes enhancement of gene expression for HOXA-10 . Transfection experiments using the HOXA-10 upstream regulatory sequence containing two putative vitamin D response elements (VDRE) into luciferase reporter constructs have revealed an activation of the reporter gene by vitamin D treatment, suggesting vitamin D can regulate HOXA-10 gene expression through direct interaction of vitamin D/ receptor complex at the promoter site. Furthermore, electrophoretic mobility shift assays clearly demonstrate that VDR binds to the HOXA-10 VDRE in the presence of 1,25- (OH) 2 D 3 . Overall, these studies suggest that vitamin D upregulates HOXA-10 expression via VDR/VDRE interaction in the HOXA-10 regulatory region. In summary, the influence of vitamin D on fertility likely occurs through regulation of uterine cell differentiation by the direct transcriptional activation of HOXA-10.

The process of decidualization is common to both mice and humans, although stromal cells in the human uterus undergo decidualization during the receptive phase (secretory phase) of each menstrual cycle in the absence of an embryo. Since Hoxa-10 is expressed in the receptive uterus and undergoes expressional alteration during decidualization or with endometrial pathogenesis (such as endometriosis), unraveling the downstream signaling pathway mediated by Hoxa-10 is clearly an interesting area of research that requires in-depth future investigation. It is not possible to use humans to study embryo–uterine interactions in early pregnancy. Mouse models serve to provide mechanistic approaches in defining a molecular basis of implantation/decidualization. Physiological functions of specific factors can be examined more mechanistically by manipulation of gene expression. Thus, the research using mice as an animal model will provide valuable information to better understand the intricacies of human pregnancy. Despite significant progress in IVF technology, the pregnancy success rate remains low, primarily due to higher incidence of unexplained pregnancy loss. Failure of pre-implantation embryo development, implantation, and placentation are major determinants for pregnancy loss. Basic research to better understand these events will help alleviate problems of infertility.

Hoxa-10 is a member of the homeobox or Hox multigene family of transcription factors ( Satokata et al., 1995 ). This gene family shares a highly conserved 61-amino-acid sequence element called the homeobox that encodes for helix-turn-helix DNA-binding domain ( Krumlauf, 1994 ). In the mammalian system, four Hox clusters (a–d) have been identified in different chromosomes, and they evolved through gene duplication ( Krumlauf, 1994 ). In general, genes localized at the 3′-end of each cluster generally undergo activation first during early embryogenesis in the anterior region, while genes at the 5′-end of the clusters express late to the posterior regions of the developing embryo ( McGinnis and Krumlauf, 1992 ; Krumlauf, 1994 ). Abdominal B ( AdbB ) is the most 5′-end gene within the Drosophila homeotic complex. In mammals, several AbdB -like genes have been identified, and they are located at the 5-ends of the Hox a, c, and d clusters ( Benson et al., 1995 ). Hoxa-10 is an AbdB -like Hox gene\expressed in the developing genitourinary system in vertebrates ( Dolle et al., 1991 ; Izpisua-Belmonte et al., 1991 ), and in the adult uterus during pregnancy ( Benson et al., 1996 ). The role of Hoxa-10 in development has been defined by gene targeting experiments ( Rijli et al., 1995 ; Satokata et al., 1995 ). Null mutation of Hoxa-10 in mice causes female infertility, which is primarily due to defective implantation and decidualization. Hoxa-10 mutant mice exhibit partial oviductal transformation of the proximal uterus, although this is shown to be unrelated to the fertility defects ( Benson et al., 1996 ). Studies have revealed that a small percentage (~40%) of Hoxa-10 −/− mice can initiate the implantation reaction. This is consistent with the observed normal expression of several implantation related genes, such as leukemia inhibitory factor ( LIF ), heparin-binding EGF ( HB-EGF ), amphiregulin , and cyclooxygenase-1 ( COX-1 ) in the uterine epithelium of Hoxa10 −/− mice ( Lim et al., 1999 ). In this regard, it is interesting to note that Hoxa10 expression is normal in Lif knockout uterus ( Daikoku et al., 2004 ), which exhibits no sign of embryo–uterine interaction ( Stewart et al., 1992 ). Overall, these studies suggest that uterine deletion of Hoxa-10 do not completely antagonize the uterine receptivity in order to induce blastocyst attachment with the uterine luminal epithelium. Previous studies have identified that Hoxa-10 is primarily a P 4 -regulated gene in the uterine endometrium ( Ma et al., 1998 ; Taylor et al., 1998 ; Lim et al., 1999 ; Godbole et al., 2007 ). In case of mice, Hoxa-10 is upregulated in the uterine stroma by P 4 in a protein synthesis-independent fashion, and this upregulation can be inhibited by progesterone receptor (PR) antagonist RU-486, suggesting PR’s requirement for this induction ( Ma et al., 1998 ). A similar observation in respect of hormonal regulation of Hoxa-10 and its antagonism by P4/PR signaling blocker was also reported in vitro with the human endometrial stromal cells ( Taylor et al., 1998 ). Consistent with these observations, studies have also shown that Hoxa-10 is regulated in the uterus during early pregnancy and its expression is primarily associated with cellular proliferation ( Huet-Hudson et al., 1989 ; Satokata et al., 1995 ; Daikoku et al., 2004 ). For example, Hoxa-10 is expressed on Days 1 and 2 of pregnancy in the luminal and glandular epithelium, expands to the stroma on Day 3, and becomes restricted to the stroma on Day 4. Following implantation by Day 5, a dramatic upregulation of this gene occurs in stromal cells undergoing decidual transformation surrounding the implanting blastocyst. On Days 6–8, the expression progressively expands to the decidualizing stroma on both mesometrial and antimesometrial poles of the implantation site ( Satokata et al., 1995 ; Daikoku et al., 2004 ). In this regard, gene knockout studies have shown that cellular proliferation within the stroma is severely compromised in the Hoxa-10 −/− mice in response to P 4 and E 2 , although epithelial cell proliferation is unaffected in response to E 2 . Consistent with this notion, certain uterine genes (prostaglandin receptors EP3 and EP4 ) that are regulated by P 4 appeared to be aberrantly expressed in the uterine stroma of Hoxa-10 −/− mice ( Lim et al., 1999 ). In humans, HOXA-10 expression in endometrial epithelial and stromal cells increases during the mid-secretory phase of the menstrual cycle, corresponding to the window of implantation ( Taylor et al., 1998 ; Gui et al., 1999 ). In nonhuman primates (bonnet monkeys), HOXA-10 protein expression appears to be different in the functionalis and basalis zones of the endometrium during the luteal phase ( Godbole et al., 2007 ). Further studies revealed that this gene is primarily controlled by P 4 , and the signals generated by the implanting embryo suggest that HOXA-10 also plays an important role in early pregnancy of the nonhuman primates. Overall, these studies suggest that the endometrial expression of Hoxa-10 plays an important role in the control of uterine receptivity, implantation, and decidualization ( Satokata et al., 1995 ; Benson et al., 1996 ; Lim et al., 1999 ). It has been well recognized that as a transcriptional factor, Hoxa-10 can influence a host of uterine genes under the direction of P 4 . In this regard, recent microarray analysis of P 4 -responsive genes in the uteri of ovariectomized Hoxa-10 null versus wild-type mice shows that many genes, including cell-cycle inhibitory genes p15 and p57, are upregulated in the null mice, implicating a negative impact on cell proliferation in the absence of Hoxa-10 ( Yao et al., 2003 ). Similarly, studies have shown that the downregulation of Hoxa-10 is correlated with the upregulation of p57 during the progression of differentiation of human endometrial stromal cells in vitro, suggesting p57 acting as a downstream regulator of Hoxa-10 presumably controls the exit of cell-cycle activity ( Qian et al., 2005 ). In this regard, it is also worth mentioning that there is a class of cyclins: cyclin G1 and cyclin G2, which normally participate as inhibitory cyclin in the cell-cycle circuitry, undergo aberrant upregulation in the Hoxa-10 −/− uteri ( Yue et al., 2005 ), suggesting that cyclin G isoforms negatively influence cell proliferation. For example, on the day of uterine receptivity (Day 4), our in situ hybridization results show that these cyclins are dramatically induced in uterine epithelial and stromal cells of Hoxa-10 mutant mice as compared to wild-type littermates ( Fig. 1 ). These results suggest that Hoxa-10 is a key player for the regulation of cyclin G’s in the uterus. The role of cyclin G isoforms in uterine biology is not yet known. The generation of cyclin G1 null mice has been reported ( Kimura et al., 2001 ; Jensen et al., 2003 ); these mice survive to adulthood and display reduced tumor susceptibility due to increased tumor suppressive function of p53. However, the reproductive phenotype of these null mice has not yet been reported, and we are currently exploring this possibility. To our knowledge, no report of cyclin G2 deletion in mice is available. Previously we reported that cyclin D3 expression follows the pattern of stromal cell proliferation and differentiation with the onset of decidualization ( Tan et al., 2002 ). Additionally, we have shown that the expression of cyclin D3 and p21, dramatically repressed in Hoxa-10 −/− uteri after application of a deciduogenic stimulus ( Das et al., 1999 ; Rahman et al., 2006 ), indicating that impaired stromal cell proliferation/differentiation is a potential cause for the defective decidualization in the null mice. Recently, a similar notion is also revealed after the suppression of Hoxa-10 during the progression of human endometrial stromal cell decidualization ( Lu et al., 2008 ). Overall, these results suggest that the Hoxa-10 mutation elicits defects that are relevant to the alteration of stromal cell responsiveness to P 4 , leading to the implantation and decidualization defects. Recent chromatin immunoprecipitation studies ( Sun et al., 2009 ) provide evidence that Hoxa-10 is a direct regulator for the control of p / CAF gene in human endometrial stromal cells. Studies revealed that HOXA-10 interacts with the p / CAF promoter comprising three consecutive TTAT elements for its ability to suppress p / CAF gene expression. P/CAF, a histone acetyltransferase, commonly mediates gene transcriptional regulation through interaction with p300/CBP and various other regulators. In this regard, the control of p/CAF-mediated physical interaction and functional modification of PTEN is particularly interesting ( Okumura et al., 2006 ). Furthermore, it is worth mentioning that conditional deletion of PTEN has been implicated in relation to the onset of endometrial cancer in mice ( Daikoku et al., 2008 ). However, the physiologic significance of HOXA-10/p/CAF/ PTEN signaling in uterine decidualization at the site of implantation remains unknown.