Yeon Sun Kim

Egr1 is rapidly and transiently induced by estrogen and bisphenol A via activation of nuclear estrogen receptor-dependent ERK1/2 pathway in the uterus.

Coordinate actions of ovarian estrogen (E2) and progesterone (P4) via their own receptors are critical for establishing uterine receptivity for embryo implantation in the uterus. E2 regulates expression of an array of genes to mediate its major actions on heterogeneous uterine cell types. Here we have investigated regulatory mechanism(s) of E2 and bisphenol A (BPA), an endocrine disruptor with potent estrogenic activity on expression of early growth response 1 (Egr1), a zinc finger transcription factor that regulates cell growth, differentiation and apoptosis in the uterus. Egr1 was rapidly and transiently induced by E2 and BPA mainly in stromal cells via nuclear estrogen receptor (ER)-ERK1/2 pathway. ICI 182,780, an ER antagonist, effectively inhibited their actions on EGR1 expression following ERK1/2 phosphorylation. Administration of pharmacological inhibitors for ERK1/2, but not AKT significantly blocked EGR1 expression induced by E2 and BPA. P4 effectively dampened action(s) of E2 and BPA on Egr1 expression via nuclear progesterone receptor. Its antagonistic effects were partially interfered with RU486 pretreatment. Interestingly, EGR1 is specifically induced in stromal cells surrounding implanting blastocyst. Collectively, our results show that through nuclear ER-dependent ERK1/2 phosphorylation, not only E2 but also endocrine disruptors with estrogenic activity such as BPA rapidly and transiently induce Egr1 which may be important for embryo implantation and decidualization in mouse uterus.

Integrative Analyses of Uterine Transcriptome and MicroRNAome Reveal Compromised LIF-STAT3 Signaling and Progesterone Response in the Endometrium of Patients with Recurrent/Repeated Implantation Failure (RIF).

Intimate two-way interactions between the implantation-competent blastocyst and receptive uterus are prerequisite for successful embryo implantation. In humans, recurrent/repeated implantation failure (RIF) may occur due to altered uterine receptivity with aberrant gene expression in the endometrium as well as genetic defects in embryos. Several studies have been performed to understand dynamic changes of uterine transcriptome during menstrual cycles in humans. However, uterine transcriptome of the patients with RIF has not been clearly investigated yet. Here we show that several signaling pathways as well as many genes and microRNAs are dysregulated in the endometrium of patients with RIF (RIFE). Whereas unsupervised hierarchical clustering showed that overall mRNA and microRNA profiles of RIFE were similar to those of endometria of healthy women, many genes were significantly dysregulated in RIFE (cut off at 1.5 fold change). The majority (~75%) of differentially expressed genes in RIFE including S100 calcium binding protein P (S100P), Chemokine (C-X-C motif) ligand 13 (CXCL13) and SIX homeobox 1 (SIX1) were down-regulated, suggesting that reduced uterine expression of these genes is associated with RIF. Gene Set Enrichment analyses (GSEA) for mRNA microarrays revealed that various signaling pathways including Leukemia inhibitory factor (LIF) signaling and a P4 response were dysregulated in RIFE although expression levels of Estrogen receptor α (ERα) and Progesterone receptor (PR) were not significantly altered in RIFE. Furthermore, expression and phosphorylation of Signal transducer and activator of transcription 3 (STAT3) are reduced and a gene set associated with Janus kinase (JAK)-STAT signaling pathway is systemically down-regulated in these patients. Pairwise analyses of microRNA arrays with prediction of dysregulated microRNAs based on mRNA expression datasets demonstrated that 6 microRNAs are aberrantly regulated in RIFE. Collectively, we here suggest that dysregulation of several major signaling pathways and genes critical for uterine biology and embryo implantation may lead to uterine abnormalities in patients with RIF.

Deficiency in DGCR8-dependent canonical microRNAs causes infertility due to multiple abnormalities during uterine development in mice

DGCR8 is an RNA-binding protein that interacts with DROSHA to produce pre-microRNA in the nucleus, while DICER generates not only mature microRNA, but also endogenous small interfering RNAs in the cytoplasm. Here, we produced Dgcr8 conditional knock-out mice using progesterone receptor (PR)-Cre (Dgcr8d/d) and demonstrated that canonical microRNAs dependent on the DROSHA-DGCR8 complex are required for uterine development as well as female fertility in mice. Adult Dgcr8d/d females neither underwent regular reproductive cycles nor produced pups, whereas administration of exogenous gonadotropins induced normal ovulation in these mice. Interestingly, immune cells associated with acute inflammation aberrantly infiltrated into reproductive organs of pregnant Dgcr8d/d mice. Regarding uterine development, multiple uterine abnormalities were noticeable at 4 weeks of age when PR is significantly increased, and the severity of these deformities increased over time. Gland formation and myometrial layers were significantly reduced, and the stromal cell compartment did not expand and became atrophic during uterine development in these mice. These results were consistent with aberrantly reduced stromal cell proliferation and completely failed decidualization. Collectively, we suggest that DGCR8-dependent canonical microRNAs are essential for uterine development and physiological processes such as proper immune modulation, reproductive cycle, and steroid hormone responsiveness in mice.

Oestrogen-induced expression of decay accelerating factor is spatiotemporally antagonised by progesterone–progesterone receptor signalling in mouse uterus

Decay accelerating factor (DAF) is upregulated in the fetoplacental trophoblast, which protects the fetus from maternal complement injury. DAF was found to be downregulated in the endometrium of patients with repeated implantation failure. Thus, we examined the molecular mechanisms of DAF expression regulation by ovarian steroid hormones in the mouse uterus. Immunofluorescence staining demonstrated its exclusive localisation in the apical region of the epithelium in the uterus. Oestrogen (E2) significantly induced Daf mRNA in a time-dependent manner. Progesterone (P4) did not have any significant effect on Daf expression; however, it negatively modulated E2-induced DAF expression and RU486 effectively interfered with the inhibitory action of P4 in the uterus. During early pregnancy DAF was higher on Day 1 of pregnancy, but significantly decreased from Day 3, which is consistent with its E2-dependent regulation. Interestingly, DAF expression seemed to be influenced by the implanting blastocyst on Day 5 and it was gradually increased during preimplantation embryo development with peak levels at blastocyst stages. We demonstrated that E2-dependent DAF expression is antagonised by P4-progesterone receptor signalling in the uterine epithelium. Spatiotemporal regulation of DAF in the uterus and preimplantation embryos suggest that DAF functions as an immune modulator for embryo implantation and early pregnancy in mice.

PLGA nanoparticles with multiple modes are a biologically safe nanocarrier for mammalian development and their offspring

Nano-sized particles (NPs) of various materials have been extensively used as therapeutic and diagnostic agents, drug delivery systems, and biomedical devices. However, the biological impacts of NP exposure during early embryogenesis on following development and next generations have not been investigated. Here, we demonstrated that polylactic-co-glycolic acid (PLGA)-NPs were not toxic and did not perturb development of preimplantation mouse embryos in vitro. Moreover, subsequent fetal development in vivo after embryo transfer proceeded normally and healthy pups were born without any genetic aberrations, suggesting biosafety of PLGA-NPs during developmental processes. TRITC-labeled PLGA-NPs, named TRITC nano-tracer (TnT) were used to visualize the successful delivery of the NPs into sperms, oocytes and early embryos. Various molecular markers for early embryogenesis demonstrated that TnT treatment at various developmental stages did not compromise embryo development to the blastocyst. mRNA-Seq analyses reinforced that TnT treatment did not significantly affect mRNA landscapes of blastocysts which undergo embryo implantation critical for following developmental processes. Moreover, when 2-cell embryos exposed to TnT were transferred into pseudopregnant recipients, healthy offspring were born without any distinct morphologic and chromosomal abnormalities. TnT treatment did not affect the sex ratio of the exposed embryos after birth. When mated with male mice, female mice that were exposed to TnT during early embryogenesis produced a comparable number of pups as control females. Furthermore, the phenotypes of the offspring of mice experienced TnT at their early life clearly demonstrated that TnT did not elicit any negative transgenerational effects on mammalian development.