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In mice, the conditional knockout strategy using the Cre-loxP system is useful for various types of research. The Cre mouse line with progesterone receptor promoter (PgrCre ) has been widely used to produce specific uterine gene-deficient mice, but in the Cre line, endogenous Pgr gene is replaced by Cre recombinase gene, which makes the breeding of homozygous mice (PgrCre/Cre ) difficult because they are infertile. Yang et al. (2013, https://10.1016/j.cell.2013.04.017) reported the generation of another PgriresCre mouse line that still has endogenous Pgr gene, and they inserted Cre recombinase downstream of the Pgr gene via an internal ribosome entry site (IRES). It is possible that this new PgriresCre line would be useful for uterine research as the mice can be bred as homozygotes (PgriresCre/iresCre ). Herein, we confirmed the PgriresCre mice effectively directed recombination in the female reproductive tract and was capable of genetic alteration in the endometrium that enables the studies of its uterine function. Our findings demonstrate that the new PgriresCre mouse line is also useful for the generation of uterine-specific knockout mice. The findings using PgriresCre mouse will contribute to the understanding of reproductive systems and diseases in humans and domestic animals.

Reproduction is the biological process that produces new living individuals and essential for the continuity of species. In mammalian reproduction, the uterus plays a pivotal role as the organ that supports the development of offspring. The main subjects of this chapter are the development and structure of the uterus. To fully make sense of the overall structure of the uterus, the process through which urogenital systems arise must be understood. Thus, we start this chapter with embryology of the uterus. Furthermore, organ structure often dictates the function, and organ function dictates the structure. This bidirectional causality is particularly evident in the human uterus: The human uterus changes in morphology dramatically in response to endocrine cues. On the other hand, the anatomy of the uterus by design dictates its physiologic function. Thus, the aim of this chapter is to provide the foundation for the following chapters through the description of embryology, anatomy, and physiology of the uterus.

During female mammal reproductive tract development, epithelial cells of the lower Müllerian duct are committed to become stratified squamous epithelium of vagina and ectocervix, when the expression of ΔNp63 transcription factor is induced by mesenchymal cells. The absence of ΔNp63 expression leads to adenosis, the putative precursor of vaginal adenocarcinoma. Our previous studies with genetically engineered mouse models have established that fibroblast growth factor (FGF)/mitogen-activated protein kinase (MAPK), bone morphogenetic protein (BMP)/SMAD, and activin A/runt related transcription factor 1 (RUNX1) signaling pathways are independently required for ΔNp63 expression in Müllerian duct epithelium (MDE). Here we report that sine oculis homeobox homolog 1 (SIX1) plays a critical role in the activation of ΔNp63 locus in MDE as a downstream transcription factor of mesenchymal signals. In mouse developing reproductive tract, SIX1 expression was restricted to MDE of the future cervix and vagina. SIX1 expression was totally absent in SMAD4 null MDE and was reduced in RUNX1 null and FGFR2 null MDE, indicating that SIX1 is under the control of vaginal mesenchymal factors, BMP4, activin A and FGF7/10. Furthermore, Six1, Runx1 and Smad4 gene-dose-dependently activated ΔNp63 expression in MDE within vaginal fornix. Using a mouse model of diethylstilbestrol (DES)-associated vaginal adenosis, we found DES action through epithelial estrogen receptor α (ESR1) down-regulates SIX1 and RUNX1 in MDE within the vaginal fornix. This study establishes that the vaginal/ectocervical cell fate of MDE is regulated by a collaboration of multiple transcription factors including SMAD4, SIX1 and RUNX1, and the down-regulation of these key transcription factors leads to vaginal adenosis.Author SummaryIn embryogenesis, differentiation fate of cells is specified through constant communication between neighboring cells. In this study, we investigated the molecular mechanism of epithelial cell fate commitment in the lower female reproductive organs utilizing mouse genetic models. The cell fate of epithelial cells in the uterus, cervix and vagina is directed by signaling from mesenchymal cells. We demonstrated that within the epithelial cells of the developing vagina, signals from mesenchymal cells are integrated into activities of transcription factors including SMAD4, RUNX1 and SIX1, which dose-dependently co-operate in the determination of vaginal epithelial cell fate. Disruption of these processes alters the cell fate from vaginal to uterine epithelium, resulting in a condition called vaginal adenosis, a putative precursor of vaginal adenocarcinoma. Women exposed to diethylstilbestrol (DES) in the womb have about 40 times the risk of developing vaginal adenocarcinoma. We determined that developmental exposure to DES induces vaginal adenosis by repressing SIX1 and RUNX1 through ESR1 in the epithelial cells. This discovery enhances the understanding of how early-life events, such as exposure to endocrine disruptors, causes vaginal adenosis, and thus may contribute to the prevention and therapeutic treatment of idiopathic vaginal adenocarcinoma.

Cell fate of lower Mullerian duct epithelium (MDE), to become uterine or vaginal epithelium, is determined by the absence or presence of ΔNp63 expression, respectively. Previously, we showed that SMAD4 and runt-related transcription factor 1 (RUNX1) were independently required for MDE to express ΔNp63. Here, we report that vaginal mesenchyme directs vaginal epithelial cell fate in MDE through paracrine activation of fibroblast growth factor (FGF) receptor-MAPK pathway. In the developing reproductive tract, FGF7 and FGF10 were enriched in vaginal mesenchyme, whereas FGF receptor 2IIIb was expressed in epithelia of both the uterus and vagina. When Fgfr2 was inactivated, vaginal MDE underwent uterine cell fate, and this differentiation defect was corrected by activation of MEK-ERK pathway. In vitro, FGF10 in combination with bone morphogenetic protein 4 and activin A (ActA) was sufficient to induce ΔNp63 in MDE, and ActA was essential for induction of RUNX1 through SMAD-independent pathways. Accordingly, inhibition of type 1 receptors for activin in neonatal mice induced uterine differentiation in vaginal epithelium by down-regulating RUNX1, whereas conditional deletion of Smad2 and Smad3 had no effect on vaginal epithelial differentiation. In conclusion, vaginal epithelial cell fate in MDE is induced by FGF7/10-MAPK, bone morphogenetic protein 4-SMAD, and ActA-RUNX1 pathway activities, and the disruption in any one of these pathways results in conversion from vaginal to uterine epithelial cell fate.

The ovary is the main secretory source of progestin and estrogen and is indispensable to the maintenance of all events of pregnancy in mice. The purpose of this study was to control all processes of pregnancy in mice, from embryo implantation to parturition, without ovaries. The ovaries were removed before embryo implantation, and a single injection of medroxyprogesterone acetate (MPA) was given. Embryo implantation was induced by leukemia inhibitory factor, which can substitute 17β-estradiol (E2). Continuous exposure to E2was necessary at mid-pregnancy, when placentation was completed. All mice sustained pregnancy without ovaries before parturition, which was initiated by the removal of E2and MPA. Murine pregnancy is a complicated process involving embryo implantation, placentation, and parturition. Complete control of pregnancy was achieved with the simple treatment of MPA and E2after induction of embryo implantation. Here, time-dependent events in the uterus during pregnancy could be realized without the ovaries, because the initiation of each event could be stringently controlled by hormonal treatments.

Leukemia inhibitory factor (LIF) is essential for embryo implantation in mice and plays an important role in other mammals including humans. Intraperitoneal (i.p.) injections with anti-LIF antibody (7.5 µg/g body weight, 3 times) between D3 (D1 = day of vaginal plug detection) and D4 effectively blocked embryo implantation; complete inhibition was achieved in C57BL/6J mice, and implantation was dramatically reduced in ICR mice (reduced to 27%). Normal rabbit IgG used as the control did not disturb embryo implantation. Anti-LIF antibody was localized not only in the stroma, but also in the luminal epithelium and the glandular lumen after i.p. injections. Growth-arrested blastocysts were recovered from the uterus without any implantation sites in both strains. Blastocysts made contact with the LE on the antimesometrial side; however, uterine stromal cells did not undergo secondary decidual reaction, and the uterine lumen was open, even at D7. Several regions of decidualization in ICR mice treated with anti-LIF antibody were smaller than those of the control, and development of blastocysts was delayed. The expression of LIF-regulated genes, such as immune-responsive gene-1 and insulin-like growth factor binding protein-3, was significantly decreased in C57BL/6J mice treated with anti-LIF antibody compared with the control, but not in ICR mice. The present study demonstrated that simple ip injections of an antibody are sufficient to block one of the important factors involved in embryo implantation in mice, and this method should also be easily applicable to the investigation of other factors involved in implantation.

Mutation of the tumor suppressor Pten often leads to tumorigenesis in various organs including the uterus. We previously showed that Pten deletion in the mouse uterus using a Pgr-Cre driver (Ptenf/fPgrCre/+) results in rapid development of endometrial carcinoma (EMC) with full penetration. We also reported that Pten deletion in the stroma and myometrium using Amhr2-Cre failed to initiate EMC. Since the Ptenf/fPgrCre/+ uterine epithelium was primarily affected by tumorigenesis despite its loss in both the epithelium and stroma, we wanted to know if Pten deletion in epithelia alone will induce tumorigenesis. We found that mice with uterine epithelial loss of Pten under a Ltf-iCre driver (Ptenf/f/LtfCre/+) develop uterine complex atypical hyperplasia (CAH), but rarely EMC even at 6 months of age. We observed that Ptenf/fPgrCre/+ uteri exhibit a unique population of cytokeratin 5 (CK5) and transformation related protein 63 (p63)-positive epithelial cells; these cells mark stratified epithelia and squamous differentiation. In contrast, Ptenf/fLtfCre/+ hyperplastic epithelia do not undergo stratification, but extensive epithelial cell apoptosis. This increased apoptosis is associated with elevation of TGFβ levels and activation of downstream effectors, SMAD2/3 in the uterine stroma. Our results suggest that stromal PTEN via TGFβ signaling restrains epithelial cell transformation from hyperplasia to carcinoma. In conclusion, this study, using tissue-specific deletion of Pten, highlights the epithelial-mesenchymal cross-talk in the genesis of endometrial carcinoma.

To determine the effect of diabetes on reproductive performance, two kinds of diabetes mice, i.e., KK/TaJcl mice with Type-II diabetes and Streptozotocin-induced diabetes mice with Type-I diabetes, were used in this study. Particular attention was paid to uterine natural killer (uNK) cells and placental growth factor (PlGF). The number of fetuses, the fetal and placental weights in both diabetes mice were significantly decreased when compared to controls. Surprisingly, uNK cells in both diabetes mice persisted in the metrial gland even at the term of pregnancy. Although PlGF expression in both diabetes mice was significantly decreased, PlGF protein did not change. These results show that diabetes condition affects reproductive performance, particularly uNK cell behavior, but not PlGF production.

Glioblastoma multiforme (GBM) is the most malignant brain tumor and resistant to cell senescence. Through proteomic screening we have identified a novel type of kinase, tentatively named, SSK1 (Super Signaling Kinase-1) that could connect cellular metabolism to cell growth. Depletion of SSK1 induced cellular senescence and diminished GBM cell growth in vitro and in a xenograft tumor model. The senescence caused by SSK1 depletion was accompanied by the induction of p21WAF1/CIP1, a critical inducer of cell senescence. Importantly, p21WAF1/CIP1 induction was independent of p53, reactive oxygen species (ROS) status, and DNA damage response pathways. Critically, the kinase activity of SSK1 is indispensable for GBM to suppress the induction of p21WAF1/CIP1 and escape senescence. Through chemical library screening, we identified a small molecule, which inhibited SSK1 activity in vitro and in vivo. Treatment of SSK1 inhibitor increased p21WAF1/CIP1 and triggered GBM senescence. Our data reveal a novel p21WAF1/CIP1 regulatory pathway mediated by SSK1, which is essential for GBM to suppress senescence, and suggest a novel therapy for their treatment, pharmacologically targeting SSK1.

Transgenic animal models are valuable for studying gene function in various tissue compartments. Mice with conditional deletion of genes in the uterus using the Cre-loxP system serve as powerful tools to study uterine biology. The uterus is comprised of 3 major tissue types: myometrium, stroma, and epithelium. Proliferation and differentiation in each uterine cell type are differentially regulated by ovarian hormones, resulting in spatiotemporal control of gene expression. Therefore, examining gene function in each uterine tissue type will provide more meaningful information regarding uterine biology during pregnancy and disease states. Although currently available Cre mouse lines have been very useful in exploring functions of specific genes in uterine biology, overlapping expression of these Cre lines in more than 1 tissue type and in other reproductive organs sometimes makes interpretation of results difficult. In this article, we report the generation of a new iCre knock-in mouse line, in which iCre is expressed from endogenous lactoferrin (Ltf) promoter. Ltf-iCre mice primarily direct recombination in the uterine epithelium in adult females and in immature females after estrogen treatment. These mice will allow for specific interrogation of gene function in the mature uterine epithelium, providing a helpful tool to uncover important aspects of uterine biology.

Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) is expressed in many organs, including female reproductive organs, and is a stem cell marker in the stomach and intestinal epithelium, hair follicles, and ovarian surface epithelium. Despite ongoing studies, the definitive physiological functions of Lgr5 remain unclear. We utilized mice with conditional deletion of Lgr5 (Lgr5d/d) in the female reproductive organs by progesterone receptor-Cre (PgrCre) to determine Lgr5's functions during pregnancy. Only 30% of plugged Lgr5d/d females delivered live pups, and their litter sizes were lower. We found that pregnancy failure in Lgr5d/d females was due to insufficient ovarian progesterone (P4) secretion that compromised decidualization, terminating pregnancy. The drop in P4 levels was reflected in elevated levels of P4-metabolizing enzyme 20α-hydroxysteroid dehydrogenase in corpora lutea (CL) inactivated of Lgr5. Of interest, P4 supplementation rescued decidualization failure and supported pregnancy to full term in Lgr5d/d females. These results provide strong evidence that Lgr5 is critical to normal CL function, unveiling a new role of LGR5 in the ovary.—Sun, X., Terakawa, J., Clevers, H., Barker, N., Daikoku, T., Dey, S. K. Ovarian LGR5 is critical for successful pregnancy.

This study aimed to clarify the mechanism of the spacing of murine embryos along the metrial and anti-metrial (MA) axis of the uterus using our newly developed experimental model. The model mice were produced by keeping mice in the supine position from the pre-implantation to implantation period. The starting points and periods of restraint of the mice in the supine position were set variously during the peri-implantation. Then, the position of the embryo was evaluated morphologically. In only one group (set in the instrument from the second day of pregnancy, Day 2, to Day 5), strong disruption of embryo spacing along the MA axis was observed. On the other hand, there was little abnormality in embryo positioning in the groups that were treated from Day 3 to Day 5 or from Day 3 to Day 6. These results suggested that determination of the position of the embryo in the MA axis is not related to duration of the experiment (2 days or 3 days), but is related to the starting time-point of the experiment, at Day 2 or Day 3. In conclusion, the period between Days 2 and 3 is critical for determination of the position of the embryo along the MA axis.

Agrin is the heparan sulfate proteoglycan (HSPG) that is well known as the molecule that aggregates acetylcholine receptor (AChR) through muscle specific kinase (MuSK) and rapsyn at neuromuscular junctions. HSPGs are spatiotemporally expressed in embryonic and maternal tissues during implantation. The present study examined the role of agrin in the mouse embryo using leukemia inhibitory factor (LIF)-deficient mice, which show complete sterility. Agrin was detected widely in the cytoplasm of uterine luminal epithelial cells at the third day of pregnancy (Day 3) and Day 4. At Day 5, agrin moved to the apical surface of the luminal epithelium. This migration was not found in LIF-deficient mice. AChR was also found in the apical surface of the uterine epithelium at Day 4 and Day 5 in normal mice. LIF-deficient mice did not show this pattern of expression. Only nAChR b1 subunit mRNA was increased at Day 5 in normal mice. Furthermore, acetylcholinesterase was active in the uterine stroma of normal mice throughout the implantation period and was exclusively active in the uterine epithelium at Day 4. Taken together, agrin signaling was activated in the uterus during embryo implantation in the mice. Here, we suggest that the agrin pathway is involved in closure of the uterine epithelium toward placentation.

Endometrial cancer is a common gynecological malignancy, causing approximately 8,000 deaths each year in the United States. Underlying causes of endometrial cancer are not clearly understood, and treatment options for patients with advanced stages are limited. Animal models that spontaneously develop cancer are powerful tools for studying the mechanism of cancer initiation and progression and for developing treatment strategies. We previously created a mouse model of endometrial cancer in which the floxed phosphatase and tensin homologue (Pten) gene was deleted by Cre expression under the control of a progesterone receptor (PR) promoter. Pten is a tumor suppressor with the highest frequency of mutation in human endometrial cancer. In the PtenloxP/loxP/PR-Cre mouse model, uterine deletion of Pten resulted in complex atypical hyperplasia of the uterine epithelium by three weeks of age with cancer occurring by one month. As PR is expressed in the epithelium, stroma, and myometrium compartments, the question remains whether Pten deletion in the uterine epithelium alone contributes to endometrial cancer since human endometrial cancer is derived from epithelial cells. To address this question, we used lactoferrin-iCre (Ltf-Cre) mice to specifically delete Pten in the uterine epithelium, since lactoferrin is restricted to the epithelium of adult mice. We found that PtenloxP/loxP/Ltf-Cre mice develop complex atypical hyperplasia in uterine epithelium by three months of age, whereas uteri from immature mice are normal. These results demonstrate that Pten deletion specifically in the uterine epithelium can contribute to toward endometrial cancer in mice. This mouse model will help us to study in detail the initiation and progression of endometrial cancer. This study was supported in parts by grants from NIH-P01-CA-77839 (S. K. D). Citation Format: Takiko Daikoku, Jumpei Terakawa, Sudhansu K. Dey. A novel mouse model of endometrial cancer: epithelial specific Pten deletion with lactoferrin-iCre. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2289. doi:10.1158/1538-7445.AM2015-2289