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In vitro decidualisation of human endometrial stromal cells is enhanced by seminal fluid extracellular vesicles.

ABSTRACT: Extracellular vesicles are highly abundant in seminal fluids and have a known role enhancing sperm function. Clinical pregnancy rates after IVF treatment are improved after female exposure to seminal fluid. Seminal fluid extracellular vesicles (SF-EVs) are candidate enhancers, however, whether SF-EVs interact with cells from the endometrium and modulate the implantation processes is unknown. Here, we investigated whether SF-EVs interact with endometrial stromal cells (ESCs) and enhance decidualisation, a requisite for implantation. SF-EVs, isolated from human seminal fluid (n = 11) by ultracentrifugation, were characterised by nanoparticle tracking analysis and Western blotting, and purified using size exclusion chromatography. Non-decidualised and decidualised primary ESCs (n = 5) were then treated with SF-EVs. Binding of bio-maleimide-labelled SF-EVs was detected by flow cytometry and fluorescence microscopy. Prolactin and IGFBP-1 protein levels in culture media were also analysed after single and multiple SF-EV exposure. SF-EVs size ranged from 50 to 300 nm, and they expressed exosomal markers (ALIX, SYNTENIN-1, CD9 and CD81). SF-EVs bound to non-decidualised and decidualised ESCs at similar levels. ESCs prolactin secretion was increased after single (p = 0.0044) and multiple (p = 0.0021) SF-EV exposure. No differences were found in IGFBP-1 protein levels. In conclusion, SF-EVs enhance in vitro ESC decidualisation and increase secretion of prolactin, an essential hormone in implantation. This elucidates a novel role of SF-EVs on endometrial receptivity. Abbreviations: ECACC: European Collection of Authenticated Cell Cultures; ESCs: endometrial stromal cells; EVs: extracellular vesicles; FCS: foetal calf serum; HRP: horse-radish peroxidase; IFNγ: interferon-gamma; IGF: insulin-like growth factor; IGFBP-1: insulin-like growth factor binding protein 1; IVF: in vitro fertilisation; MVB: multivesicular bodies; NTA: nanoparticle tracking analysis; PRLR-/-: homozygous prolactin receptor knockout; RT: room temperature; SF-EVs: seminal fluid extracellular vesicles; STR: short tandem repeat; TGFβ: transforming growth factor β; uNK: uterine natural killer.

SUBMITTER: Rodriguez-Caro H

PROVIDER: S-EPMC6352950 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The uterine response to the presence of embryos is poorly understood in the domestic dog (Canis familiaris). The intimate embryo-maternal cross-talk, which begins following the hatching of blastocysts and embryo attachment leads to strong structural and functional remodelling of the uterus. A part of this process is decidualisation, comprising morphological and biochemical changes that result in formation of maternal stroma-derived decidual cells. These are an integral part of the canine placenta materna, which together with the maternal vascular endothelium are the only cells of the canine endotheliochorial placenta able to resist trophoblast invasion. These cells are also the only ones within the canine placenta expressing the progesterone receptor (PGR). Understanding the decidualisation process thus appears essential for understanding canine reproductive physiology.Here, we investigated the capability of canine uterine stromal cells to decidualise in vitro, thereby serving as a canine model of decidualisation. A dbcAMP-mediated approach was chosen during a time course of 24 - 72 h. Tissue material from six (n?=?6) healthy, dioestric bitches was used (approximately 2 weeks after ovulation). Cells were characterized by differential staining, nearly 100 % of which were vimentin-positive. Scanning and transmission electron microscope analyses were applied, and morphological changes were recorded with a live cell imaging microscope. Expression of several decidualisation markers was investigated.The in vitro cultured stromal cells acquired characteristics of decidual cells when incubated with 0.5 mM dbcAMP for 72 h. Their shape changed from elongated to rounded, while ultrastructural analysis revealed higher numbers of mitochondria and secretory follicles, and an increased proliferation rate. Elevated expression levels of IGF1, IGF2, PRLR and ER? were observed in decidualised cells; PRL and ER? remained mostly below the detection limit, while PGR remained unaffected. The expression of smooth muscle ? actin (?SMA), another decidualisation marker, was strongly induced. Among prostaglandin system members, levels of COX2 (PTGS2) and of PGE2-synthase (PTGES) were upregulated. Expression of the PGE2 receptors, PTGER2 and PTGER4, was clearly detectable.An in vitro decidualisation model with canine uterine stromal cells was successfully established, allowing future, more detailed studies to be undertaken on the underlying molecular and endocrine mechanisms of canine decidualisation.

Project description:In pregnancy, the decidualised endometrium expresses high levels of prorenin and other genes of the renin-angiotensin system (RAS) pathway. In this study we aimed to determined if the RAS was present in endometrial stromal cells and if decidualisation upregulated the expression of prorenin, the prorenin receptor ((P)RR) and associated RAS pathways. Immortalised human endometrial stromal cells (HESCs) can be stimulated to decidualise by combined treatment with medroxyprogesterone acetate (MPA), 17?-estradiol (E2) and cAMP (MPA-mix) or with 5-aza-2'-deoxycytidine (AZA), a global demethylating agent.HESCs were incubated for 10 days with one of the following treatments: vehicle, MPA-mix, a combination of medroxyprogesterone acetate (MPA) and estradiol-17? alone, or AZA. Messenger RNA abundance and protein levels of prorenin (REN), the (P)RR (ATP6AP2), angiotensinogen (AGT), angiotensin converting enzyme (ACE), angiotensin II type 1 receptor (AGTR1), vascular endothelial growth factor (VEGF), and plasminogen activator inhibitor-1 (PAI-1) were measured by real-time PCR and ELISA's, respectively. Promyelocytic zinc finger (PLZF) and phospho-inositol-3 kinase (PIK3R1) mRNA abundances were also measured.HESCs expressed the prorenin receptor (ATP6AP2), REN, AGT, ACE and low levels of AGTR1. MPA-mix and AZA stimulated expression of REN. Prorenin protein secretion was increased in MPA-mix treated HESCs. E2?+?MPA had no effect on any RAS genes. MPA-mix treatment was associated with increased VEGF (VEGFA) and PAI-1 (SERPINE1) mRNA and VEGF protein.An endometrial prorenin receptor/renin angiotensin system is activated by decidualisation. Since (P)RR is abundant, the increase in prorenin secretion could have stimulated VEGF A and SERPINE1 expression via Ang II, as both ACE and AGTR1 are present, or by Ang II independent pathways. Activation of the RAS in human endometrium with decidualisation, through stimulation of VEGF expression and secretion, could be critical in establishing an adequate blood supply to the developing maternal placental vascular bed.

Project description:STUDY QUESTION: Do extracellular vesicles (EVs) from human Fallopian tubes exert an influence on early embryo development in vitro? SUMMARY ANSWER: Human Fallopian tube EVs carrying miRNAs increase murine embryo viability in vitro. WHAT IS KNOWN ALREADY: Oviductal EVs (oEVs) are recently identified key players in embryo-oviduct interactions that contribute to successful pregnancy in vivo. Their absence in current in vitro systems may partly explain the suboptimal embryo development observed, therefore further knowledge is needed about their impact on early embryos. STUDY DESIGN, SIZE, DURATION: The oEVs were isolated from the luminal fluid of human Fallopian tubes using ultracentrifugation. We cocultured oEVs with murine two-cell embryos until the blastocyst stage. The study was conducted between August 2021 and July 2022. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 23 premenopausal women were recruited for Fallopian-tubes collection, and the oEVs were isolated. The micro RNA (miRNA) contents were detected using high-throughput sequencing and their target genes and effects were analyzed. After in vitro culture with or without oEVs, the blastocyst and hatching rates were recorded. Furthermore, for the blastocysts formed, we assessed the total cell number, inner cell mass proportion, reactive oxygen species (ROS) level, number of apoptotic cells and mRNA expression levels of genes involved in development. MAIN RESULTS AND THE ROLE OF CHANCE: EVs were successfully isolated from the human Fallopian tubal fluid and the concentrations were evaluated. A total of 79 known miRNAs were identified from eight samples that had been sequenced, all involved in various biological processes. The blastocyst rate, hatching rate, as well as total cell number of blastocysts were significantly increased in the oEVs-treated groups (p < 0.05 versus untreated), while the proportion of inner cell mass showed no significant difference between groups. ROS levels and apoptotic cell proportions were decreased in the oEVs-treated groups (p < 0.05 versus untreated). The genes, Actin-Related Protein 3 (Actr3), Eomesodermin (Eomes), and Wnt Family Member 3A (Wnt3a) were upregulated in blastocysts in the oEVs-treated group.

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In vitro decidualisation of human endometrial stromal cells is enhanced by seminal fluid extracellular vesicles.

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