*2.3. Hormone Signaling*

Uterine function is primarily regulated by estrogen and progesterone, which modulate gene expression of luminal and glandular epithelium and stromal cells. These ovarian hormones guide the structural and functional remodeling occurring during decidualization. Estrogen receptor (ER) exists in two forms, ERα and ERβ with distinct biological function, both expressed in the endometrium. ERα is essential for implantation since ERα knockout mice display endometrial hypoplasia and are infertile [74]; mice knockout for ERβ present normal endometrium and appear fertile, suggesting that ERβ may be involved in other aspects of endometrial function [75–77]. During the proliferative phase, high levels of estrogen induce proliferation of the epithelial, stromal, and vascular endothelial cells [78,79]. Indeed, activated ERα induces proliferation of human epithelial cells and decidualization in stromal cells through rapid non-genomic activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway [80,81]. In addition to this rapid activity, estrogen increases epithelial proliferation by inducing insulin-like growth factor 1 (IGF1). IGF1 is expressed and secreted by the stroma, and by binding its receptor IGF1R in the epithelium, induces the phosphoinositide 3-kinase (PI3K)/ serine/threonine protein kinase (AKT) pathway leading to proliferation [82–84]. Other

known targets of estrogen in the endometrium are fibroblast growth factor (FGF)-9, CCAAT enhancer binding protein beta (C/EBPβ) and Mucin 1 (MUC1). FGF-9 is expressed at high levels in the stromal compartment of the endometrium during the late proliferative phase; in vitro FGF-9 stimulates stromal cell proliferation, and expression of FGF-9 in such cells is induced upon 17β-estradiol stimulation [85], suggesting that in vivo estrogen may induce proliferation of stromal cells through the up-regulation of FGF-9. Estrogen-induced proliferation of endometrial cells is also mediated by C/EBPβ, whose pro-proliferative action is exerted on both the endometrial epithelium and stroma through regulation of cyclin-dependent kinases involved in G2 to M transition of mitosis [86]. Estrogen also regulates the expression of the glycoprotein MUC1, which is expressed on the surface of the luminal epithelium to create a protective barrier that has to be removed at implantation to allow embryo attachment [87]. Beyond its activity on cell proliferation, estrogen also induces endometrial expression of leukemia inhibitory factor (LIF), an interleukin-6 family cytokine whose central role for successful implantation and decidualization has been widely reported [88,89] and discussed later in this review. During the proliferative phase, estrogen induces progesterone receptor (PR) in endometrial cells through ERα to determine progesterone responsiveness during the luteal phase, and in turn PR inhibits ERα expression in a negative feedback crucial for endometrial function [90]. Progesterone increases during the secretory phase of the menstrual cycle, inducing decidualization and thus opening the WOI and remains elevated if pregnancy occurs [91]. The effects of progesterone in endometrial cells are mediated by PR, which exists in two isoforms, PR-A and PR-B, transcribed from two promoters of the same gene. Deletion of either PR-A or PR-B demonstrates specific roles of each PR isoform in mediating progesterone actions on the murine uterus. In PR-B knockout mice, progesterone effects mediated by PR-A are sufficient for a normal uterine function, since implantation, pregnancy, and parturition are normal in these mice [92]. On the contrary, in PR-A-knockout mice progesterone actions mediated by PR-B lead to increased hyperplasia of the endometrial epithelium and inflammation, and no decidualization in the endometrial stroma [93]. Taken together these data indicate that PR-A is critical for implantation, and that PR-B is involved in endometrial differentiation. Female mice knockout for both PR-A and PR-B are infertile, showing severely reduced or absent ovulation, uterine hyperplasia, absence of decidualization, severely limited mammary gland development, and an impaired sexual behavior [94]. After progesterone binding, PR activates a series of signal transductions, involving both genomic and non-genomic pathways. The non-genomic response is rapid and based on the interaction with c-Src kinase to induce the pro-proliferative ERK/MAPK and AKT pathways, important for peri-implantation stromal proliferation [90,95,96]. The genomic action of PR involves its translocation into the nucleus and modulation of gene expression [90]. In the uterine epithelium, progesterone promotes the expression of Indian hedgehog (IHH), which in turn induces up-regulation of stromal chicken ovalbumin upstream promoter-transcription factor II (COUPTFII) that regulates stromal bone morphogenetic protein 2 (BMP2) and consequently the decidualization response of the stromal compartment [97–100]. Progesterone-mediated induction of IHH is also responsible for the down-regulation of MUC1 [98]. As for the effect of progesterone on decidualization, it has been demonstrated that progesterone stimulation induces heart and neural crest derivatives expressed 2 (HAND2), a transcription factor, whose down-regulation in mouse and human fibroblast cells is associated to reduction of decidualization markers [101].

The genomic response to progesterone action also regulates the expression of transcription factors of the homeobox family [102]. Homeobox protein-A10 and -A11 (HOXA10, HOXA11), are expressed in stromal and glandular compartments of the endometrium throughout the menstrual cycle and are both essential for pregnancy, since their deletion in mice results in implantation defects [103–105]. Both HOXA10 and HOXA11 have a role in decidualization [104,106]. HOXA10 positively regulates the expression of the decidual marker Insulin Growth Factor Binding Protein 1 (IGFBP1) [107], while HOXA11 normally function as a repressor of the decidual marker prolactin (PRL) gene, however in cooperation with FOXO1A it induces its 3-fold increase [104]. In vitro models have greatly contributed in understanding the role of progesterone in decidualization [108,109]. Treatment of endometrial organ

cultures or endometrial derived stromal cells with progesterone induces expression of PRL [109,110], but with higher efficiency if steroid hormones are used in combination with cAMP [111,112]. cAMP alone can induce decidualization of human endometrial stromal cells (HESCs) but for few days only [113–115], since for the stabilization of the process it is necessary the presence of both cAMP and progesterone [112].

In addition to the steroid hormones produced by the ovary, other hormones are involved in the establishment of pregnancy, among which one of the most studied is the chorionic gonadotropin (CG). CG is produced by the trophectoderm of the blastocyst and is one of the main players involved in endometrium–embryo crosstalk at the time of implantation. The ovaries respond to CG, which acts as an agonist of LH, by maintaining the corpus luteum, thus producing the progesterone necessary for the establishment and progression of pregnancy [116]. The responses of the endometrium are multiple, including the inhibition of apoptosis, which usually occurs at the end of the menstrual cycle, by activating anti-apoptotic genes as B-cell lymphoma 2 (BCL-2) [117,118], and the induction of the decidualization process [118–120]. Both epithelial and stromal cells possess the LH/CG receptor (LHCGR), a seven transmembrane G protein-coupled receptor, which shows the highest expression during the secretory phase of the menstrual cycle [119,121,122]. Endometrial epithelial cells respond to CG by expressing cyclooxygenase-2 (COX2) and prostaglandin E synthase (PGES), through the activation of extracellular signal-regulated protein kinases 1/2 (Erk1/2) signaling pathway. The increased production of prostaglandin E2 (PGE2) [122–124] induces cAMP in endometrial stromal cells and promotes their decidualization [124,125]. COX-derived PGE2 plays an important role in the increase of endometrial vascular permeability, which characterizes the inflammatory reaction typical of implantation [126,127]. In endometrial stromal cells CG activates Erk1/2 signaling pathway, thus increasing the expression of the PR and regulating the expression of genes controlling endometrial receptivity [121]. Moreover, in primates, endometrial stromal cells respond to CG and progesterone by activating Notch receptor 1 (NOTCH1) pathway, as discussed later. NOTCH1 induces the expression of α-smooth muscle actin (α-SMA), which positively regulates remodeling of cytoskeleton and the initial changes typical of the decidualization process [128]. Subsequently, a decrease in CG and NOTCH1 levels is necessary for the completion of decidualization, which is accompanied by an increase in the expression of insulin-like growth factor binding protein-1 (IGFBP1) and prolactin (PRL), markers of decidualization [129–131], and a downregulation of LHCGR [120,132–134].
