Long-term, hormone-responsive organoid cultures of human endometrium in a chemically defined medium

In humans, the endometrium, the uterine mucosal lining, undergoes dynamic changes throughout the menstrual cycle and pregnancy. Despite the importance of the endometrium as the site of implantation and nutritional support for the conceptus, there are no long-term culture systems that recapitulate endometrial function in vitro. We adapted conditions used to establish human adult stem-cell-derived organoid cultures to generate three-dimensional cultures of normal and decidualized human endometrium. These organoids expand long-term, are genetically stable and differentiate following treatment with reproductive hormones. Single cells from both endometrium and decidua can generate a fully functional organoid. Transcript analysis confirmed great similarity between organoids and the primary tissue of origin. On exposure to pregnancy signals, endometrial organoids develop characteristics of early pregnancy. We also derived organoids from malignant endometrium, and so provide a foundation to study common diseases, such as endometriosis and endometrial cancer, as well as the physiology of early gestation.This work was supported by the Medical Research Council (MR/L020041/1), the Centre for Trophoblast Research, University of Cambridge and the Wellcome Trust (RG60992). M.Y.T. has received funding from the E.U. 7th Framework Programme for research, technological development and demonstration under grant agreement no PIEF-GA-2013-629785. J.H. was supported by a Wellcome Trust vacation scholarship. B.-K.K. is supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society (101241/Z/13/Z) and receives a core support grant from the Wellcome Trust and MRC to the WT-MRC Cambridge Stem Cell Institute

Trophoblast organoids as a model for maternal-fetal interactions during human placentation.

The placenta is the extraembryonic organ that supports the fetus during intrauterine life. Although placental dysfunction results in major disorders of pregnancy with immediate and lifelong consequences for the mother and child, our knowledge of the human placenta is limited owing to a lack of functional experimental models1. After implantation, the trophectoderm of the blastocyst rapidly proliferates and generates the trophoblast, the unique cell type of the placenta. In vivo, proliferative villous cytotrophoblast cells differentiate into two main sub-populations: syncytiotrophoblast, the multinucleated epithelium of the villi responsible for nutrient exchange and hormone production, and extravillous trophoblast cells, which anchor the placenta to the maternal decidua and transform the maternal spiral arteries2. Here we describe the generation of long-term, genetically stable organoid cultures of trophoblast that can differentiate into both syncytiotrophoblast and extravillous trophoblast. We used human leukocyte antigen (HLA) typing to confirm that the organoids were derived from the fetus, and verified their identities against four trophoblast-specific criteria3. The cultures organize into villous-like structures, and we detected the secretion of placental-specific peptides and hormones, including human chorionic gonadotropin (hCG), growth differentiation factor 15 (GDF15) and pregnancy-specific glycoprotein (PSG) by mass spectrometry. The organoids also differentiate into HLA-G+ extravillous trophoblast cells, which vigorously invade in three-dimensional cultures. Analysis of the methylome reveals that the organoids closely resemble normal first trimester placentas. This organoid model will be transformative for studying human placental development and for investigating trophoblast interactions with the local and systemic maternal environment.Centre for Trophoblast Reearch Royal Society Dorothy Hodgkin Fellowship Marie Curie Intra-European Fellowshi

Embryonic Diapause Is Conserved across Mammals

Embryonic diapause (ED) is a temporary arrest of embryo development and is characterized by delayed implantation in the uterus. ED occurs in blastocysts of less than 2% of mammalian species, including the mouse (Mus musculus). If ED were an evolutionarily conserved phenomenon, then it should be inducible in blastocysts of normally non-diapausing mammals, such as domestic species. To prove this hypothesis, we examined whether blastocysts from domestic sheep (Ovis aries) could enter into diapause following their transfer into mouse uteri in which diapause conditions were induced. Sheep blastocysts entered into diapause, as demonstrated by growth arrest, viability maintenance and their ED-specific pattern of gene expression. Seven days after transfer, diapausing ovine blastocysts were able to resume growth in vitro and, after transfer to surrogate ewe recipients, to develop into normal lambs. The finding that non-diapausing ovine embryos can enter into diapause implies that this phenomenon is phylogenetically conserved and not secondarily acquired by embryos of diapausing species. Our study questions the current model of independent evolution of ED in different mammalian orders

Localized induction of gene expression in embryonic stem cell aggregates using holographic optical tweezers to create biochemical gradients

Three-dimensional (3D) cell models that mimic the structure and function of native tissues are enabling more detailed study of physiological and pathological mechanisms in vitro. We have previously demonstrated the ability to build and manipulate 3D multicellular microscopic structures using holographic optical tweezers (HOTs). Here, we show the construction of a precisely patterned 3D microenvironment and biochemical gradient model consisting of mouse embryoid bodies (mEBs) and polymer microparticles loaded with retinoic acid (RA), embedded in a hydrogel. We demonstrate discrete, zonal expression of the RA-inducible protein Stra8 within mEBs in response to release of RA from polymer microparticles, corresponding directly to the defined 3D positioning of the microparticles using HOTs. These results demonstrate the ability of this technology to create chemical microgradients at definable length scales and to elicit, with fidelity and precision, specific biological responses. This technique can be used in the study of in vitro microenvironments to enable new insights on 3D cell models, their cellular assembly, and the delivery of drug or biochemical molecules for engineering and interrogation of functional and morphogenic responses

Development of sheep androgenetic embryos is boosted following transfer of male pronuclei into androgenetic hemizygotes.

ABSTRACTAndrogenetic embryos are useful model for investigating the contribution of the paternal genometo embryonic development. Little work has been done with androgenetic embryo productionin domestic animals. The aim of this study was the production of diploid androgeneticsheep embryos. In vitro matured sheep oocytes were enucleated and fertilized in vitro;parthenogenetic and normally fertilized embryos were also produced as a control. Fifteenhours after in vitro fertilization (IVF), presumptive zygotes were centrifuged and scored forthe number of pronucleus. IVF, parthenogenetic, and androgenetic embryos (haploid, diploid,and triploid) were cultured in SOFaa medium with bovine serum albumin (BSA). The proportionof oocytes with polyspermic fertilization increased linearly with increasing spermconcentration. After IVF, there was no significant difference in early cleavage and morula formationrates between the groups, while there was a significant difference on blastocyst developmentbetween IVF, parthenogenetic, and androgenetic embryos, the last ones displayingpoor developmental potential (IVF, parthenogenetic, and haploid, diploid, and triploidandrogenetic embryos: 43%, 38%, 0%, 2%, and 2%, respectively). In order to boost androgeneticembryonic development, we produced diploid androgenetic embryos through pronucleartransfer. Single pronuclei were aspirated with a bevelled pipette from haploid or diploidembryos and transferred into the perivitelline space of other haploid embryos, and the zygoteswere reconstructed by electrofusion. Fusion rates approached 100%. Pronuclear transfersignificantly increased blastocyst development (IVF, parthenogenetic, androgenetic:Diploid into Haploid, and Haploid into Haploid: 42%, 42%, 19%, and 3%, respectively); intriguingly,the Haploid Diploid group showed the highest development to blastocyst stage.The main findings of our study are: (1) sheep androgenetic embryos display poor developmentalability compared with IVF and parthenogenetic embryos; (2) diploid androgenetic embryosproduced by pronuclear exchange developed in higher proportion to blastocyst stage,particularly in the Diploid–Haploid group. In conclusion, pronuclear transfer is an effectivemethod to produce sheep androgenetic blastocysts.[...

High levels of anandamide, an endogenous cannabinoid, block the growth of sheep preimplantation embryos by inducing apoptosis and reversible arrest of cell proliferation.

BACKGROUND: The process of implantation is mediated by various molecules, one of which is anandamide (AEA), a lipid signalling ligand belonging to the family of endocannabinoids. AEA exerts its effects on implantation by binding to the Type 1 Cannabinoid Receptor (CB1-R), expressed in both blastocysts and uterus. We wanted to know whether the endocannabinoid signalling system was present also in the sheep reproductive tract and which kind of effect(s) AEA had on the development of sheep blastocysts in vitro. METHODS: We analysed the expression and activity of the endocannabinoid system in sheep reproductive tracts and blastocysts. Hatched sheep blastocysts were then exposed to AEA and its effect(s) were determined by TUNEL assay and by measuring the rate of necrosis and 5-bromo-deoxyuridine incorporation. RESULTS: We show that the AEA signalling system is present in sheep and that high concentrations of AEA induce apoptosis and inhibit cell proliferation via a CB1-R-dependent mechanism. Indeed, AEA effects were blocked when sheep blastocysts were cultured in the presence of the CB1-R antagonist SR161417A. Moreover, AEA inhibition of cell proliferation was reversible, as arrested embryos resumed a normal growth rate upon AEA removal from the medium. CONCLUSIONS: Our results suggest that disturbed regulation of AEA signalling via CB1-R may be associated with pregnancy failure. AEA could lower the quality of blastocysts by inducing apoptosis and inhibiting cell proliferation, thus making them incompetent for implantation

High levels of anandamide, an endogenous cannabinoid, block the growth of sheep preimplantation embryos by inducing apoptosis and reversible arrest of cell proliferation

High levels of anandamide, an endogenous cannabinoid, blockthe growth of sheep preimplantation embryos by inducingapoptosis and reversible arrest of cell proliferationM.Y. Turco1, K. Matsukawa1, M. Czernik1, V. Gasperi1, N. Battista1, L. Della Salda1,P.A. Scapolo1, P. Loi1, M. Maccarrone1,2,3† and G. Ptak1,3†1Department of Comparative Biomedical Sciences, University of Teramo 64100, Teramo, Italy; 2European Centre for Brain Research(CERC)/IRCCS S. Lucia Foundation, 00143 Rome, Italy3Correspondence address. Tel: þ39-0861-266837; Fax: þ39-0861-411285; E-mail: [email protected] (G.P.)/Tel: þ39-0861-266875;Fax: þ39-0861-266877; E-mail: [email protected] (M.M.)BACKGROUND: The process of implantation is mediated by various molecules, one of which is anandamide (AEA), alipid signalling ligand belonging to the family of endocannabinoids. AEA exerts its effects on implantation by bindingto the Type 1 Cannabinoid Receptor (CB1-R), expressed in both blastocysts and uterus. We wanted to know whetherthe endocannabinoid signalling system was present also in the sheep reproductive tract and which kind of effect(s)AEA had on the development of sheep blastocysts in vitro. METHODS: We analysed the expression and activity ofthe endocannabinoid system in sheep reproductive tracts and blastocysts. Hatched sheep blastocysts were thenexposed to AEA and its effect(s) were determined by TUNEL assay and by measuring the rate of necrosis and5-bromo-deoxyuridine incorporation. RESULTS: We show that the AEA signalling system is present in sheep andthat high concentrations of AEA induce apoptosis and inhibit cell proliferation via a CB1-R-dependent mechanism.Indeed, AEA effects were blocked when sheep blastocysts were cultured in the presence of the CB1-R antagonistSR161417A. Moreover, AEA inhibition of cell proliferation was reversible, as arrested embryos resumed a normalgrowth rate upon AEA removal from the medium. CONCLUSIONS: Our results suggest that disturbed regulationof AEA signalling via CB1-R may be associated with pregnancy failure. AEA could lower the quality of blastocystsby inducing apoptosis and inhibiting cell proliferation, thus making them incompetent for implantation.Keywords: anandamide; endocannabinoids; embryo development; implantation; sheepIntroductionSuccessful cross-talk between the embryo and receptive uterusmust take place to allow implantation and establish pregnancy.This process involves a sequence of finely regulated events thatultimately lead to adhesion and intimate contact between theblastocyst trophectoderm (TE) and the maternal endometrium.The cross-talk is mediated by many factors, whose exact rolesand pathways have not yet been fully determined. One of thesefactors is N-arachidonoyl-ethanolamine (anandamide, AEA), along-chain, unsaturated fatty acid-derivative that belong to afamily of lipid mediators described as the endocannabinoidsystem (Mechoulam, 2002; De Petrocellis et al., 2004; Bariet al., 2006). This endogenous ligand was identified as aresult of studies on the effects of D9-tetrahydrocannabinol(THC), the active constituent of marijuana, as AEA and THCshare their main target, i.e. the G-protein-coupled receptor,Type 1 Cannabinoid Receptor (CB1-R) (Mastuda et al., 1990).The use of illicit drugs, particularly marijuana, amongwomen has raised serious concerns about their effects on pregnancy(Sherwood et al., 1999; Fergusson et al., 2002). A significantnumber of females exposed to THC have reportedunsuccessful pregnancies due to failure of implantation orspontaneous abortion (Maccarrone et al., 2000a,b), lowerinfant birth weight (Fergusson et al., 2002) and cognitive deficitsof the offspring (Fried et al., 2003). These data and otherssuggest that the endocannabinoid system is operative in thereproductive apparatus (fo[...