Uterine-specific Ezh2 deletion enhances stromal cell senescence and impairs placentation, resulting in pregnancy loss.

Maternal uterine remodeling facilitates embryo implantation, stromal cell decidualization and placentation, and perturbation of these processes may cause pregnancy loss. Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that epigenetically represses gene transcription; loss of uterine EZH2 affects endometrial physiology and induces infertility. We utilized a uterine Ezh2 conditional knockout (cKO) mouse to determine EZH2's role in pregnancy progression. Despite normal fertilization and implantation, embryo resorption occurred mid-gestation in Ezh2cKO mice, accompanied by compromised decidualization and placentation. Western blot analysis revealed Ezh2-deficient stromal cells have reduced amounts of the histone methylation mark H3K27me3, causing upregulation of senescence markers p21 and p16 and indicating that enhanced stromal cell senescence likely impairs decidualization. Placentas from Ezh2cKO dams on gestation day (GD) 12 show architectural defects, including mislocalization of spongiotrophoblasts and reduced vascularization. In summary, uterine Ezh2 loss impairs decidualization, increases decidual senescence, and alters trophoblast differentiation, leading to pregnancy loss.

Identification and characterization of novel abdominal and pelvic brown adipose depots in mice.

Brown adipose tissue (BAT) generates heat through non-shivering thermogenesis, and increasing BAT amounts or activity could facilitate obesity treatment and provide metabolic benefits. In mice, BAT has been reported in perirenal, thoracic and cranial sites. Here, we describe new pelvic and lower abdominal BAT depots located around the urethra, internal reproductive and urinary tract organs and major lower pelvic blood vessels, as well as between adjacent muscles where the upper hind leg meets the abdominal cavity. Immunohistochemical, western blot and PCR analyses revealed that these tissues expressed BAT markers such as uncoupling protein 1 (UCP1) and CIDEA, but not white adipose markers, and ß3-adrenergic stimulation increased UCP1 amounts, a classic characteristic of BAT tissue. The newly identified BAT stores contained extensive sympathetic innervation with high mitochondrial density and multilocular lipid droplets similar to interscapular BAT. BAT repositories were present and functional neonatally, and showed developmental changes between the neonatal and adult periods. In summary, several new depots showing classical BAT characteristics are reported and characterized in the lower abdominal/pelvic region of mice. These BAT stores are likely significant metabolic regulators in the mouse and some data suggests that similar BAT depots may also exist in humans.

Regulation of AKT Signaling in Mouse Uterus.

17ß-estradiol (E2) treatment of ovariectomized adult mice stimulates the uterine PI3K-AKT signaling pathway and epithelial proliferation through estrogen receptor 1 (ESR1). However, epithelial proliferation occurs independently of E2/ESR1 signaling in neonatal uteri. Similarly, estrogen-independent uterine epithelial proliferation is seen in adulthood in mice lacking Ezh2, critical for histone methylation, and in wild-type (WT) mice treated neonatally with estrogen. The role of AKT in estrogen-independent uterine epithelial proliferation was the focus of this study. Expression of the catalytically active phosphorylated form of AKT (p-AKT) and epithelial proliferation were high in estrogen receptor 1 knockout and WT mice at postnatal day 6, when E2 concentrations were low, indicating that neither ESR1 nor E2 are essential for p-AKT expression and epithelial proliferation in these mice. However, p-AKT levels and proliferation remained estrogen responsive in preweaning WT mice. Expression of p-AKT and proliferation were both high in uterine luminal epithelium of mice estrogenized neonatally and ovariectomized during adulthood. Increased expression of phosphorylated (inactive) EZH2 was also observed. Consistent with this, Ezh2 conditional knockout mice show ovary-independent uterine epithelial proliferation and high epithelial p-AKT. Thus, adult p-AKT expression is constitutive and E2/ESR1 independent in both model systems. Finally, E2-induced p-AKT expression and normal uterine proliferation did not occur in mice lacking membrane (m)ESR1, indicating a key role for membrane ESR1 in AKT activation. These findings emphasize the importance of AKT activation in promoting uterine epithelial proliferation even when that proliferation is not E2/ESR1 dependent and further indicate that p-AKT can be uncoupled from E2/ESR1 signaling in several experimental scenarios.

Role of nuclear and membrane estrogen signaling pathways in the male and female reproductive tract.

Estrogen signaling through the main estrogen receptor, estrogen receptor 1 (ESR1; also known as ERα), is essential for normal female and male reproductive function. Historically, studies of estrogen action have focused on the classical genomic pathway. Although this is clearly the major pathway for steroid hormone actions, these hormones also signal through rapid non-classical effects involving cell membrane actions. Reports of rapid effects of estrogens extend for more than half a century, but recent results have expanded understanding of the identity, structure, function and overall importance of membrane receptors in estrogen responses. Key findings in this field were the immunohistochemical detection of ESR1 in cell membranes and demonstration that a portion of newly synthesized ESR1 is routed to the membrane by palmitoylation. These receptors in the membrane can then signal through protein kinases and other mechanisms following ligand binding to alter cell function. Another crucial advance in the field was development of transgenic mice expressing normal amounts of functional nuclear ESR1 (nESR1) but lacking membrane ESR1 (mESR1). Both male and female transgenic mice lacking mESR1 were infertile as adults, and both sexes had extensive reproductive abnormalities. Transgenic mice lacking mESR1 were highly protected from deleterious effects of neonatal estrogen administration, and estrogen effects on the histone methyltransferase Enhancer of Zeste homolog 2 that are mediated through mESR1 could have significant effects on epigenetic imprinting. In summary, signaling through mESR1 is essential for normal male and female reproductive function and fertility, and is a critical enabler of normal estrogen responses in vivo. Although the precise role of mESR1 in estrogen responses remains to be established, future research in this area should clarify its mechanism of action and lead to a better understanding of how mESR1 signaling works with classical genomic signaling through nESR1 to promote full estrogenic responses.

MicroRNA-145 targets Smad1 in endometrial stromal cells and regulates decidualization in rat.

Decidualization of endometrial stromal cells is the pre-requisite for the embryo implantation and establishment of pregnancy. Although known to be regulated by several factors, the process of regulation of decidualization by miRNAs is largely unknown. Previous reports suggest that the upregulated expression of miR-145 is associated with repeated implantation failure. The current study was aimed to identify and validate the role of miR-145 in regulating stromal cell decidualization and the mechanism involved therein. Expression of miR-145 was found to be downregulated during the decidualization period of early pregnancy and also in artificially induced decidualization in rat uterus. During in vitro decidualization in rat endometrial stromal cells (ESCs), the overexpression of mimic miR-145 attenuated the progression of decidualization. Biochemical marker alkaline phosphatase and protein markers (insulin-like growth factor binding protein, cyclin D3) were also suppressed in miR-145 mimic-transfected cells as compared to normal decidualized cells. Bioinformatic analysis and luciferase reporter assay confirmed that Smad1 is the direct target of miR-145. Differentiation of ESCs was inhibited in miR-145 mimic-transfected cells which occurred via downregulating the target Smad1 along with its downstream p-Smad1/5/8 and Wnt-4. Pre-treatment of ESCs with Smad1 siRNA resulted in downregulated expression of p-Smad1/5/8, Wnt-4, Cox-2, and VEGF. In addition, miR-145 overexpression resulted in the loss of angiogenic factors Cox-2, MMP-9, and VEGF, indicating suppression of the process of angiogenesis. Migration of human umbilical vein endothelial cells was also attenuated in the presence of conditioned media obtained from miR-145-transfected decidualizing cells. In conclusion, the study demonstrated the role of miR-145 in regulation of progression of decidualization which is mediated through inhibition of Smad1. KEY MESSAGES: MiR-145 expression is downregulated during decidualization in the rat uterus. Overexpression of miR-145 inhibited the decidualization progression. MiR-145 suppressed the migration and invasion of HUVECs. MiR-145 downregulated Smad1 which suppresses Smad1/5/8, Wnt-4, MMP-9, Cox-2, and VEGF.