Progesterone receptor isoform B regulates the Oxtr-Plcl2-Trpc3 pathway to suppress uterine contractility.

Uterine contractile dysfunction leads to pregnancy complications such as preterm birth and labor dystocia. In humans, it is hypothesized that progesterone receptor isoform PGR-B promotes a relaxed state of the myometrium, and PGR-A facilitates uterine contraction. This hypothesis was tested in vivo using transgenic mouse models that overexpress PGR-A or PGR-B in smooth muscle cells. Elevated PGR-B abundance results in a marked increase in gestational length compared to control mice (21.1 versus 19.1 d respectively, P < 0.05). In both ex vivo and in vivo experiments, PGR-B overexpression leads to prolonged labor, a significant decrease in uterine contractility, and a high incidence of labor dystocia. Conversely, PGR-A overexpression leads to an increase in uterine contractility without a change in gestational length. Uterine RNA sequencing at midpregnancy identified 1,174 isoform-specific downstream targets and 424 genes that are commonly regulated by both PGR isoforms. Gene signature analyses further reveal PGR-B for muscle relaxation and PGR-A being proinflammatory. Elevated PGR-B abundance reduces Oxtr and Trpc3 and increases Plcl2 expression, which manifests a genetic profile of compromised oxytocin signaling. Functionally, both endogenous PLCL2 and its paralog PLCL1 can attenuate uterine muscle cell contraction in a CRISPRa-based assay system. These findings provide in vivo support that PGR isoform levels determine distinct transcriptomic landscapes and pathways in myometrial function and labor, which may help further the understanding of abnormal uterine function in the clinical setting.

Constitutive expression of progesterone receptor isoforms promotes the development of hormone-dependent ovarian neoplasms.

Differences in the relative abundances of the progesterone receptor (PGR) isoforms PGRA and PGRB are often observed in women with reproductive tract cancers. To assess the importance of the PGR isoform ratio in the maintenance of the reproductive tract, we generated mice that overexpress PGRA or PGRB in all PGR-positive tissues. Whereas few PGRA-overexpressing mice developed reproductive tract tumors, all PGRB-overexpressing mice developed ovarian neoplasms that were derived from ovarian luteal cells. Transcriptomic analyses of the ovarian tumors from PGRB-overexpressing mice revealed enhanced AKT signaling and a gene expression signature similar to those of human ovarian and endometrial cancers. Treating PGRB-overexpressing mice with the PGR antagonist RU486 stalled tumor growth and decreased the expression of cell cycle-associated genes, indicating that tumor growth and cell proliferation were hormone dependent in addition to being isoform dependent. Analysis of the PGRB cistrome identified binding events at genes encoding proteins that are critical regulators of mitotic phase entry. This work suggests a mechanism whereby an increase in the abundance of PGRB relative to that of PGRA drives neoplasia in vivo by stimulating cell cycling.

Pinopodes: Recent advancements, current perspectives, and future directions.

Successful embryo implantation is a complex and highly regulated process involving precise synchronization between the fetal-derived trophoblast cells and maternal uterine luminal epithelium. Multiple endocrine-driven factors are important for controlling the timely receptivity of the uterus, and this complexity underscores implantation failure as a major cause of recurrent infertility associated with assisted reproductive technologies. One particular cellular structure often hypothesized to promote receptivity is the pinopode or uterodome - a hormonally regulated, large cellular protrusion on the uterine epithelial surface. Recent clinical studies associate pinopodes with favorable fertility outcomes in women, and because they are directly linked to an increase in progesterone levels, the potential utility of these hormone-regulated cell biological structures in predicting or improving implantation in a clinical setting holds promise. In this review, we aim to generate interest in pinopodes from the broader cell biology and endocrinology communities, re-examine methodologies in pinopode research, and identify priorities for future investigation of pinopode structure and function in women's reproductive health.

E-Cigarette Exposure Delays Implantation and Causes Reduced Weight Gain in Female Offspring Exposed In Utero.

Electronic nicotine delivery system (e-cigarette) use is prevalent among pregnant women as a seemingly safe alternative to traditional tobacco use, known to result in fetal developmental abnormalities and impaired fertility of male offspring. However, little is known about the effects of e-cigarette use on fertility or pregnancy outcomes. A successful pregnancy is initiated by a multitude of dynamic molecular alterations in the uterus resulting in embryo implantation at day 4.5 in the mouse. We examined whether e-cigarette exposure impairs implantation and offspring health. Pregnant C57BL/6J mice were exposed five times a week to e-cigarette vapor or sham. After 4 months, e-cigarette exposed dams exhibited a significant delay in the onset of the first litter. Furthermore, exposure of new dams in early pregnancy significantly impaired embryo implantation, as evidenced by nearly complete absence of implantation sites in e-cigarette-exposed animals at day 5.5, despite exhibiting high levels of progesterone, an indicator of pregnancy. RNA microarray from day 4.5 pseudopregnant mice revealed significant changes in the integrin, chemokine, and JAK signaling pathways. Moreover, female offspring exposed to e-cigarettes in utero exhibited a significant weight reduction at 8.5 months, whereas males exhibited a slight but nonsignificant deficiency in fertility. Thus, e-cigarette exposure in mice impairs pregnancy initiation and fetal health, suggesting that e-cigarette use by reproductive-aged women or during pregnancy should be considered with caution.

FOXO1 regulates uterine epithelial integrity and progesterone receptor expression critical for embryo implantation.

Successful embryo implantation requires a receptive endometrium. Poor uterine receptivity can account for implantation failure in women who experience recurrent pregnancy loss or multiple rounds of unsuccessful in vitro fertilization cycles. Here, we demonstrate that the transcription factor Forkhead Box O1 (FOXO1) is a critical regulator of endometrial receptivity in vivo. Uterine ablation of Foxo1 using the progesterone receptor Cre (PgrCre) mouse model resulted in infertility due to altered epithelial cell polarity and apoptosis, preventing the embryo from penetrating the luminal epithelium. Analysis of the uterine transcriptome after Foxo1 ablation identified alterations in gene expression for transcripts involved in the activation of cell invasion, molecular transport, apoptosis, ß-catenin (CTNNB1) signaling pathway, and an increase in PGR signaling. The increase of PGR signaling was due to PGR expression being retained in the uterine epithelium during the window of receptivity. Constitutive expression of epithelial PGR during this receptive period inhibited expression of FOXO1 in the nucleus of the uterine epithelium. The reciprocal expression of PGR and FOXO1 was conserved in human endometrial samples during the proliferative and secretory phase. This demonstrates that expression of FOXO1 and the loss of PGR during the window of receptivity are interrelated and critical for embryo implantation.

A mouse model engineered to conditionally express the progesterone receptor-B isoform.

Using a Rosa26 gene targeting strategy in mouse embryonic stem cells, we have generated a new transgenic mouse (Pgr-B LSL ), which is designed to conditionally express the epitope-tagged mouse progesterone receptor-B (PGR-B) isoform when crossed with a specific cre driver mouse. To functionally validate this transgenic mouse, we crossed the Pgr-B LSL mouse with the MMTV-CREA transgenic mouse to create the MMTV-CREA/Pgr-B LSL bigenic (termed PR-B:OE to denote PGR-B overexpressor). As expected, transgene-derived PGR-B protein was specifically targeted to the virgin mammary gland epithelium. At a functional level, the PR-B:OE bigenic exhibited abnormal mammary morphogenesis-dilated epithelial ducts, precocious alveologenesis and lateral side-branching, along with a prominent proliferative signature-that resulted in pregnant PR-B:OE mice unable to exhibit mammary gland terminal differentiation at parturition. Because of this developmental failure, the PR-B:OE mammary gland was incapable of producing milk resulting in early neonatal death of otherwise healthy litters. This first line of analysis demonstrates the utility of the Pgr-B LSL mouse to examine the role of the PGR-B isoform in different physiologic and pathophysiologic systems that are responsive to progesterone.

Decreased epithelial progesterone receptor A at the window of receptivity is required for preparation of the endometrium for embryo attachment.

The precise timing of progesterone signaling through its cognate receptor, the progesterone receptor (PGR), is critical for the establishment and maintenance of pregnancy. Loss of PGR expression in the murine uterine epithelium during the preimplantation period is a marker for uterine receptivity and embryo attachment. We hypothesized that the decrease in progesterone receptor A (PGRA) expression is necessary for successful embryo implantation. To test this hypothesis, a mouse model constitutively expressing PGRA (mPgrALsL/+) was generated. Expression of PGRA in all uterine compartments (Pgrcre) or uterine epithelium (Wnt7acre) resulted in infertility with defects in embryo attachment and stromal decidualization. Expression of critical PGRA target genes, indian hedgehog, and amphiregulin (Areg), was maintained through the window of receptivity while the estrogen receptor target gene, the leukemia inhibitory factor (Lif), a key regulator of embryo receptivity, was decreased. Transcriptomic and cistromic analyses of the mouse uterus at day 4.5 of pregnancy identified an altered group of genes regulating molecular transport in the control of fluid and ion levels within the uterine interstitial space. Additionally, LIF and its cognate receptor, the leukemia inhibitory factor receptor (LIFR), exhibited PGR-binding events in regions upstream of the transcriptional start sites, suggesting PGRA is inhibiting transcription at these loci. Therefore, downregulation of the PGRA isoform at the window of receptivity is necessary for the attenuation of hedgehog signaling, transcriptional activation of LIF signaling, and modulation of solutes and fluid, producing a receptive environment for the attaching embryo.

Progesterone receptor signaling in the initiation of pregnancy and preservation of a healthy uterus.

Infertility and reproductive-associated disease are global problems in the world today affecting millions of women. A successful pregnancy requires a healthy uterus ready to receive and support an implanting embryo. As an endocrine organ, the uterus is dependent on the secretions of the ovarian hormones estrogen and progesterone which signal via their cognate receptors, the estrogen and progesterone receptors. The progesterone receptor not only functions using classical nuclear receptor signaling, but also participates in non-genomic signaling at the cellular membrane. The complexity of progesterone signaling is further enhanced by the existence of multiple isoforms and post-translational regulation via kinases and transcription coregulators. This dynamic means of regulation of the progesterone receptor is evidenced in its necessary role in a successful pregnancy. Within early pregnancy, the progesterone receptor elicits activation of its target genes in a spatiotemporal manner in order to allow for successful embryo attachment and uterine decidualization. Additionally, appropriate progesterone signaling is important for the prevention of uterine disease such as endometrial cancer, endometriosis, and leiomyoma. The utilization of progesterone receptor modulators in the treatment of these devastating uterine diseases is promising. This review presents a general overview of progesterone receptor structure, function, and regulation and highlights its important role in the establishment of pregnancy and as a therapeutic target in uterine disease.

The epidermal growth factor receptor critically regulates endometrial function during early pregnancy.

Infertility and adverse gynecological outcomes such as preeclampsia and miscarriage represent significant female reproductive health concerns. The spatiotemporal expression of growth factors indicates that they play an important role in pregnancy. The goal of this study is to define the role of the ERBB family of growth factor receptors in endometrial function. Using conditional ablation in mice and siRNA in primary human endometrial stromal cells, we identified the epidermal growth factor receptor (Egfr) to be critical for endometrial function during early pregnancy. While ablation of Her2 or Erbb3 led to only a modest reduction in litter size, mice lacking Egfr expression are severely subfertile. Pregnancy demise occurred shortly after blastocyst implantation due to defects in decidualization including decreased proliferation, cell survival, differentiation and target gene expression. To place Egfr in a genetic regulatory hierarchy, transcriptome analyses was used to compare the gene signatures from mice with conditional ablation of Egfr, wingless-related MMTV integration site 4 (Wnt4) or boneless morphogenic protein 2 (Bmp2); revealing that not only are Bmp2 and Wnt4 key downstream effectors of Egfr, but they also regulate distinct physiological functions. In primary human endometrial stromal cells, marker gene expression, a novel high content image-based approach and phosphokinase array analysis were used to demonstrate that EGFR is a critical regulator of human decidualization. Furthermore, inhibition of EGFR signaling intermediaries WNK1 and AKT1S1, members identified in the kinase array and previously unreported to play a role in the endometrium, also attenuate decidualization. These results demonstrate that EGFR plays an integral role in establishing the cellular context necessary for successful pregnancy via the activation of intricate signaling and transcriptional networks, thereby providing valuable insight into potential therapeutic targets.

The progesterone receptor regulates implantation, decidualization, and glandular development via a complex paracrine signaling network.

Many women are affected by infertility and reproductive-associated disease such as endometriosis or endometrial cancer. Successful pregnancy is dependent on a healthy uterus that is fit to receive and support a fertilized embryo. The uterus is an endocrine organ, responsive to the presence of the ovarian steroid hormones, estrogen and progesterone, which activate transcription of target genes through the binding of their cognate receptors, the estrogen receptor and the progesterone receptor. Progesterone signaling has been demonstrated to be critical for the initiation and continuance of pregnancy. Through the induction of Ihh, Wnt, and Bmp pathways within the epithelial and stromal compartments of the uterus, embryo attachment and implantation occur followed by decidualization of the surrounding stroma. Furthermore, these pathways have been shown to be involved in uterine glandular development. This review highlights the integral role of uterine progesterone-mediated paracrine signaling in gland development and pregnancy.

Epithelial progesterone receptor exhibits pleiotropic roles in uterine development and function.

The ovarian steroid progesterone, acting through the progesterone receptor (PR), coordinates endometrial epithelial-stromal cell communication, which is critical for its development and function. PR expression in these cellular compartments is under tight temporal and endocrine control. Although ex vivo studies demonstrated the importance of stromal PR expression, they failed to show a role for epithelial PR in uterine function. Here, the in vivo role of PR in the uterine epithelium is defined using floxed PR (PR(f/f)) mice crossed to Wnt7a-Cre mice. Progesterone was unable to stimulate the expression of its epithelial target genes, including Ihh, in the Wnt7a-Cre(+)PR(f/-) mice. Analysis was conducted on Ihh to determine whether PR directly regulates epithelial gene transcription. ChIP-on-chip analysis identified PR binding sites in the 5'-flanking region of Ihh. Cotransfection of the proximal Ihh promoter with PR demonstrated that PR directly regulates Ihh transcription. Female Wnt7a-Cre(+)PR(f/-) mice are infertile due to defects in embryo attachment, stromal cell decidualization, and the inability to cease estrogen-induced epithelial cell proliferation. Finally, progesterone was unable to inhibit neonatal endometrial glandular development in Wnt7a-Cre(+)PR(f/-) mice. Thus, epithelial PR is necessary for the regulation of progesterone epithelial target gene expression, as well as uterine function and development.