Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801
Performing Department
Veterinary Research & Extension
Non Technical Summary
Implantation of the embryo to the uterine wall is governed by an intimate interaction between the trophoblast cells of the embryo and the receptive uterine lining of the mother. While many aspects of implantation remain unclear, there is little doubt that intercellular communications between trophoblast, endometrial epithelial and stromal cells are vital for successful implantation and establishment of pregnancy. The signals that mediate crosstalk between these various cell- types remain poorly understood. Our recent studies revealed that the steroid hormone estrogen induces the expression of the transcription factor hypoxia-inducible factor 2 alpha (HIF2a) in subluminal stromal cells of mouse uterus at the time of implantation. We observed that conditional deletion of the Hif2a gene in the uterus leads to infertility. In uteri lacking HIF2a, the blastocyst trophectoderm is closely apposed to the uterine epithelium but fails to sustain firm attachment to it, resulting in an implantation failure. Further studies using gene expression profiling revealed downregulation of Rab GTPases in HIF2a-null stromal cells. Rabs belong to a large family of GTPases and are involved in various membrane trafficking events. Interestingly, the loss of HIF2a in endometrial stromal cells affected a distinct set of Rabs that regulate secretion of extracellular vesicles (EVs), including microvesicles and exosomes.Interestingly, a recent study has shown that EVs are present in luminal fluid of the ovine uterus. Since emerging studies indicate that EVs mediate a novel mechanism of cell signaling and communication between various cell types, we hypothesize that EVs secreted by the ovine endometrial stromal cells (OESC) are involved in cell-cell communication during implantation. We have proposed two aims to address this hypothesis. In Aim 1A we will isolate EVs and determine whether EV secretion is enhanced as an adaptive response to hypoxia in a primary culture of OESC. We will then determine whether stromal EVs are taken up by ovine endometrial epithelial cells. In Aim 1B we will attenuate Hif2a expression in OESC and determine the expression of Rab GTPases in these cells. We will also monitor the secretion of EVs in the conditioned media of OESC cultures.Successful pregnancy is essential for the survival and continued development of species. It is now well recognized that many gestational complications and pregnancy losses occur at very early stages around embryo implantation. In the ewe, estimates of embryonic death loss are 20- 40%, with the majorityof the losses occurring during theperi-implantation period of pregnancy. Studies proposed in this project will help us to generate knowledge that may have a great impact on our understanding of intricate signaling mechanisms that operate between distinct cellular compartments of the uterus during early pregnancy.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
In the absence of HIF2a, the stromal cells exhibit a marked impairment in the expression of Rab11, Rab22a and Rab27a that are critical regulators of EV secretion. There is a growing appreciation that EVs, by transferring their cargo of proteins, mRNAs and microRNAs to neighboring and even distant cells, represent an important mechanism of intercellular communication. To test the hypothesis that HIF2a regulates stromal EV secretion, we will:Aim 1A: Isolate EVs and determine whether EV secretion is enhanced as an adaptive response to hypoxia in a primary culture of OESC. We will then determine whether stromal EVs are taken up by epithelial cells.Aim 1B: Attenuate Hif2a expression in OESC and determine the expression of Rab GTPases in OESC. Monitor the secretion of EVs in the conditioned media of OESC cultures.
Project Methods
Please note that theexperiments proposed in this project will employ an ovine endometrial cell line and that no live animal work, therefore, is required.Ovine endometrial stromal cells (OESC) at a confluency of 70-80% will be cultured under normoxic (20%) or hypoxic (3%) conditions. Cells will be exposed to estrogen and progesterone to induce differentiation. After 24, 48, and 72 hours of culture in the presence or absence of hypoxia, conditioned media will be harvested for EV isolation using low speed centrifugation followed by precipitation with the ExoQuick-TC (System Biosciences). Briefly, conditioned media will undergo serial centrifugation (300xg to remove cells; 10m, 200xg to remove dead cells; 10m, 10,000xg; 30m to remove cell debris, macroparticles and apoptotic bodies) prior to Exoquick precipitation. EV precipitation with the Exoquick reagent will be performed according to manufacturer's instructions. Pelleted EVs will be re-suspended in PBS. Cell counts and viability will be also determined at the time of harvest in order to account for differences in cell growth. To distinguish between microvesicles and exosomes, we will perform western blot analysis using antibodies against CD63 and heat shock protein 70 (HSP70), two well-characterized exosomal protein markers. These experiments will inform us about the presence of EVs in the conditioned media of endometrial stromal cells.We will then check whether epithelial cells can internalize stromal EVs. EVs isolated from stromal cells will be labeled using the red lipophilic fluorescent dye PKH26 according to the manufacturer's instructions and published methods. Ovine endometrial epithelial cells will be seeded on cover slips in 24-well plates and cultured for 24 hours. The cells will be then incubated with the red PKH26-labelled EVs for an additional 24 hours, after which the cells will be washed, mounted and analyzed by confocal microscopy. We expect confocal microscopy to reveal that red fluorescent vesicles are internalized by epithelial cells, indicating uptake of EVs.To determine whether Hif2a regulates the expression of Rabs, we will attenuate Hif2a expression in OESC by employing RNAi strategy. OESC will be placed in culture under normoxic and hypoxic (95% nitrogen plus 3-5% oxygen in a hypoxic incubator) conditions. Cells will be allowed to grow to reach ~80% confluency and then transfected with Hif2a siRNA and control luciferase siRNA with the Dharmafect transfection reagent using procedures that are established in the laboratory. siRNA concentrations from 100 nM to 200 nM will be used to determine the optimal concentration that produces maximal Hif2a mRNA and protein knockdown without affecting non-target transcripts such as progesterone receptor, GAPDH and 36B4. Six hours after transfection, fresh culture medium will be added and decidualization will be initiated in the stromal cells by the addition of estrogen and progesterone. OESC treated with control and test siRNAs will be harvested after 1, 2, 4, and 8 days from the time of initiation of culture. Cell lysates will be prepared and HIF2a protein levels will be assessed by western blotting. RNA will also be isolated from these cells and analyzed by qPCR for the expression of Hif2a target genes such as Rab11, Rab22a, and Rab27a.If we observe that attenuation of Hif2a expression in OESC regulates expression of genes, such as the Rabs, we will isolate conditioned media from these cultures at different times following the initiation of culture. We will investigate whether the secretion of EVs in the conditioned media of OESC is affected as a consequence of suppression of Hif2a expression. These experiments will establish whether Hif2a controls a conserved signaling pathway in the mouse and OESC.