In vitro decidualisation of canine uterine stromal cells

BACKGROUND: 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. METHODS: 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. RESULTS: 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 ERα were observed in decidualised cells; PRL and ERβ remained mostly below the detection limit, while PGR remained unaffected. The expression of smooth muscle α actin (αSMA), 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. CONCLUSION: 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12958-015-0066-4) contains supplementary material, which is available to authorized users

The IGF system in in-vitro human decidualization

Decidualization of endometrial stromal cells (ESCs) is critical for a successful pregnancy but the molecular mechanisms of the process are poorly understood. In this study, we investigated whether the insulin-like growth factor (IGF) network is involved in this cellular process. Expression kinetics of members of the IGF system was examined at both mRNA and protein levels during in-vitro decidualization of cultured human ESCs. We found a significant up-regulation of IGF-II as well as of IGF-I receptor and the A and B insulin receptor (InsR) isoforms. In addition, levels of the key adaptor proteins insulin receptor substrate 1 (IRS-1) and IRS-2 increased, suggesting a potential involvement of the IGF signalling pathway in the decidualization process. Expression of two IGF binding proteins, IGFBP-1 and IGFBP-4, which can inhibit IGF action, also increased. In order to determine whether IGF signalling was activated during decidualization, the phosphorylation status of the receptors and the adaptor proteins was estimated. Only IRS-2 was slightly phosphorylated in decidualized cells and was further activated by the addition of exogenous IGF-II. These results suggest that the IGF signalling pathway could play a crucial role in the functions of decidualized endometrial cell

Social work with older people as specialisation

peer reviewedThe alternative or noncanonical NF-kappaB pathway regulates the generation of p52-containing NF-kappaB dimers (e.g., p52/RelB) through a partial degradation (called processing) of the precursor p100 into p52. This pathway is activated by a subset of non-death TNF receptor members, which ultimately activate two kinases: NIK (NF-kappaB-Inducing Kinase) and IKKalpha (Inhibitor of kappaB Kinase alpha). These kinases create a phosphodegron for the E3 ligase SCF-beta-TrCP that covalently binds K48-linked polyubiquitin chain onto p100 prior to its proteasomal processing. The resulting p52-containing complexes translocate into the nucleus to activate target genes involved in secondary lymphoid organ development, B cell survival or in osteoclastogenesis.We describe in this chapter straightforward methods to monitor the activation of the alternative NF-kappaB pathway. These methods uncover cytosolic and nuclear biochemical modifications of key proteins of the alternative NF-kappaB pathway required prior to the transcription of NF-kappaB target genes

NIK promotes tissue destruction independently of the alternative NF-kappaB pathway through TNFR1/RIP1-induced apoptosis.

NF-kappaB-inducing kinase (NIK) is well-known for its role in promoting p100/NF-kappaB2 processing into p52, a process defined as the alternative, or non-canonical, NF-kappaB pathway. Here we reveal an unexpected new role of NIK in TNFR1-mediated RIP1-dependent apoptosis, a consequence of TNFR1 activation observed in c-IAP1/2-depleted conditions. We show that NIK stabilization, obtained by activation of the non-death TNFRs Fn14 or LTbetaR, is required for TNFalpha-mediated apoptosis. These apoptotic stimuli trigger the depletion of c-IAP1/2, the phosphorylation of RIP1 and the RIP1 kinase-dependent assembly of the RIP1/FADD/caspase-8 complex. In the absence of NIK, the phosphorylation of RIP1 and the formation of RIP1/FADD/caspase-8 complex are compromised while c-IAP1/2 depletion is unaffected. In vitro kinase assays revealed that recombinant RIP1 is a bona fide substrate of NIK. In vivo, we demonstrated the requirement of NIK pro-death function, but not the processing of its substrate p100 into p52, in a mouse model of TNFR1/LTbetaR-induced thymus involution. In addition, we also highlight a role for NIK in hepatocyte apoptosis in a mouse model of virus-induced TNFR1/RIP1-dependent liver damage. We conclude that NIK not only contributes to lymphoid organogenesis, inflammation and cell survival but also to TNFR1/RIP1-dependent cell death independently of the alternative NF-kappaB pathway.Cell Death and Differentiation advance online publication, 5 June 2015; doi:10.1038/cdd.2015.69

The effect of DNA methylation inhibitor 5-Aza-2'-deoxycytidine on human endometrial stromal cells

Decidualization, the differentiation of endometrial stromal cells is a crucial step for successful implantation of an embryo, development of the placenta and completion of pregnancy to term. Epigenetic mechanisms are thought to be strongly involved in the regulation of processes controlling implantation, placentation, organ formation and foetal growth. Recent studies suggest that decreased DNA methylation facilitates a receptive endometrium. Hence, the aim of this project was to compare the transcriptional profile changes induced by the inhibitor of DNA methylation, 5-Aza-2'-deoxycytidine (AZA) to the transcriptional changes that happen during decidualization