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Morphological, Ultrastructural, and Molecular Aspects of In Vitro Mouse Embryo Implantation on Human Endometrial Mesenchymal Stromal Cells in The Presence of Steroid Hormones as An Implantation Model.

ABSTRACT: Objective:This experimental study aimed to evaluate the effects of 17?-estradiol (E2) and progesterone (P4) on the interaction between mouse embryo and human endometrial mesenchymal stromal cells, and gene expressions related to implantation [?V and ?3 integrins, interleukin-1 receptor (IL-1R), and leukemia inhibitory factor receptor (LIFR)] using an in vitro twodimensional model. Materials and Methods:In this experimental study, the endometrial stromal cells were isolated enzymatically and mechanically, and cultured to the fourth passage. Next, their immunophenotype was confirmed by flow cytometric analysis as mesenchymal stromal cells. The cells were cultured as either the experimental group in the presence of E2 (0.3 nmol) and P4 (63.5 nmol) or control group without any hormone treatment. Mouse blastocysts were co-cultured with endometrial mesenchymal stromal cells in both groups for 48 hours. Their interaction was assessed under an inverted microscope and scanning electron microscopy (SEM). Expressions of ?V and ?3 integrins, LIFR, and IL-1R genes were analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR). Results:Similar observations were seen in both groups by light microscopy and SEM. We observed the presence of pinopode-like structures and cell secretions on the apical surfaces of endometrial mesenchymal stromal cells in both groups. The trophoblastic cells expanded and interacted with the mesenchymal monolayer cells. At the molecular level, expression of IL-1R significantly increased in the hormonal treated group compared to the control (P?0.05). Expressions of the other genes did not differ. Conclusion:This study has shown that co-culture of endometrial mesenchymal stromal cells with mouse embryo in media that contained E2 (0.3 nmol) and P4 (63.5 nmol) could effectively increase the expression of IL-1R, which is involved in embryo implantation. However, there were no significant effects on expressions of ?V and ?3 integrins, LIFR, and on the morphology and ultrastructure of endometrial mesenchymal stromal cells.

SUBMITTER: Rahimipour M

PROVIDER: S-EPMC6004996 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Morphological, Ultrastructural, and Molecular Aspects of In Vitro Mouse Embryo Implantation on Human Endometrial Mesenchymal Stromal Cells in The Presence of Steroid Hormones as An Implantation Model.

Rahimipour Marzieh M Salehnia Mojdeh M Jafarabadi Mina M

Cell journal 20180515 3

<h4>Objective</h4>This experimental study aimed to evaluate the effects of 17β-estradiol (E2) and progesterone (P4) on the interaction between mouse embryo and human endometrial mesenchymal stromal cells, and gene expressions related to implantation [αV and β3 integrins, interleukin-1 receptor (IL-1R), and leukemia inhibitory factor receptor (LIFR)] using an in vitro twodimensional model.<h4>Materials and methods</h4>In this experimental study, the endometrial stromal cells were isolated enzymat ...[more]

PMID: 29845791

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Project description:BackgroundReceptivity of the uterus is essential for embryo implantation and progression of mammalian pregnancy. Acquisition of receptivity involves major molecular and cellular changes in the endometrial lining of the uterus from a non-receptive state at ovulation, to a receptive state several days later. The precise molecular mechanisms underlying this transition and their upstream regulators remain to be fully characterized. Here, we aimed to generate a comprehensive profile of the endometrial transcriptome in the peri-ovulatory and peri-implantation states, to define the genes and gene pathways that are different between these states, and to identify new candidate upstream regulators of this transition, in the mouse.ResultsHigh throughput RNA-sequencing was utilized to identify genes and pathways expressed in the endometrium of female C57Bl/6 mice at estrus and on day 3.5 post-coitum (pc) after mating with BALB/c males (n = 3-4 biological replicates). Compared to the endometrium at estrus, 388 genes were considered differentially expressed in the endometrium on day 3.5 post-coitum. The transcriptional changes indicated substantial modulation of uterine immune and vascular systems during the pre-implantation phase, with the functional terms Angiogenesis, Chemotaxis, and Lymphangiogenesis predominating. Ingenuity Pathway Analysis software predicted the activation of several upstream regulators previously shown to be involved in the transition to receptivity including various cytokines, ovarian steroid hormones, prostaglandin E2, and vascular endothelial growth factor A. Our analysis also revealed four candidate upstream regulators that have not previously been implicated in the acquisition of uterine receptivity, with growth differentiation factor 2, lysine acetyltransferase 6 A, and N-6 adenine-specific DNA methyltransferase 1 predicted to be activated, and peptidylprolyl isomerase F predicted to be inhibited.ConclusionsThis study confirms that the transcriptome of a receptive uterus is vastly different to the non-receptive uterus and identifies several genes, regulatory pathways, and upstream drivers not previously associated with implantation. The findings will inform further research to investigate the molecular mechanisms of uterine receptivity.

Project description:Steroid receptor coactivator-3 (SRC-3; also known as NCOA3 or AIB1) is a member of the multifunctional p160/SRC family of coactivators, which also includes SRC-1 and SRC-2. Clinical and cell-based studies as well as investigations on mice have demonstrated pivotal roles for each SRC in numerous physiological and pathophysiological contexts, underscoring their functional pleiotropy. We previously demonstrated the critical involvement of SRC-2 in murine embryo implantation as well as in human endometrial stromal cell (HESC) decidualization, a cellular transformation process required for trophoblast invasion and ultimately placentation. We show here that, like SRC-2, SRC-3 is expressed in the epithelial and stromal cellular compartments of the human endometrium during the proliferative and secretory phase of the menstrual cycle as well as in cultured HESCs. We also found that SRC-3 depletion in cultured HESCs results in a significant attenuation in the induction of a wide-range of established biomarkers of decidualization, despite exposure of these cells to a deciduogenic stimulus and normal progesterone receptor expression. These molecular findings are supported at the cellular level by the inability of HESCs to morphologically transform from a stromal fibroblastoid cell to an epithelioid decidual cell when endogenous SRC-3 levels are markedly reduced. To identify genes, signaling pathways and networks that are controlled by SRC-3 and potentially important for hormone-dependent decidualization, we performed RNA-sequencing on HESCs in which SRC-3 levels were significantly reduced at the time of administering the deciduogenic stimulus. Comparing HESC controls with HESCs deficient in SRC-3, gene enrichment analysis of the differentially expressed gene set revealed an overrepresentation of genes involved in chromatin remodeling, cell proliferation/motility, and programmed cell death. These predictive bioanalytic results were confirmed by the demonstration that SRC-3 is required for the expansion, migratory and invasive activities of the HESC population, cellular properties that are required in vivo in the formation or functioning of the decidua. Collectively, our results support SRC-3 as an important coregulator in HESC decidualization. Since perturbation of normal homeostatic levels of SRC-3 is linked with common gynecological disorders diagnosed in reproductive age women, this endometrial coregulator-along with its new molecular targets described here-may open novel clinical avenues in the diagnosis and/or treatment of a non-receptive endometrium, particularly in patients presenting non-aneuploid early pregnancy loss.

Project description:PurposeDuring early embryo invasion (48 h after embryo attachment), what functional changes accompany dynamic gene expression alterations in human endometrial stromal cells?MethodIn the present study, primary human endometrial stromal cells (phESCs) were cultured. After in vitro decidualization, primary human endometrial stromal cells (phESCs) were cultured with blastocysts for 48 h. During this process, blastocysts attached and invaded the phESCs (embryo-invaded primary human endometrial stromal cells, ehESCs). We performed comprehensive transcriptomic profiling of phESCs (two replicates) and ehESCs (five replicates) and analyzed the differentially expressed gene (DEGs) sets for gene ontology (GO) terms and Kyoto encyclopaedia of genes and genomes (KEGG) pathway enrichment. To analyse potential connectivity patterns between the transcripts in these DEG sets, a protein-protein interaction (PPI) network was constructed using the STRING database.ResultsA total of 592 DEGs were identified between phESCs and ehESCs after embryo invasion. Primary human endometrial stromal cells underwent significant transcriptomic changes that occur in a stepwise fashion. Oxidative phosphorylation, mitochondrial organization, and P53 signalling pathways were significantly altered in phESCs after embryo invasion. EP300 may play a key role in regulating transcription via chromatin remodelling to facilitate the adaptive gene expression changes that occur during embryo invasion.ConclusionsOur data identify dynamic transcriptome changes that occur in endometrial stromal cells within 48 h after embryo invasion. The pathways that we found to be enriched in phESCs after embryo invasion (oxidative phosphorylation, mitochondrial organization, and P53 signalling) may represent novel mechanisms underlying embryo implantation, and may illuminate the reasons that some women experience reproductive failure.Key messagesHuman endometrial stromal cells have undergone changes in gene expression regulation and signalling pathways during the embryo invasion.Mitochondrial-oxidative phosphorylation changes in human stromal cells manifested as down-regulation of gene expression in the electron transport chain.TP53 signalling pathway and transcriptional regulator EP300 assist stromal cells to get adaptive changes during embryo invasion phase.

Dataset Information

Morphological, Ultrastructural, and Molecular Aspects of In Vitro Mouse Embryo Implantation on Human Endometrial Mesenchymal Stromal Cells in The Presence of Steroid Hormones as An Implantation Model.

Publications

Morphological, Ultrastructural, and Molecular Aspects of In Vitro Mouse Embryo Implantation on Human Endometrial Mesenchymal Stromal Cells in The Presence of Steroid Hormones as An Implantation Model.

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