scholarly journals Characterisation of Osteopontin in an In Vitro Model of Embryo Implantation

Cells ◽  
2019 ◽  
Vol 8 (5) ◽  
pp. 432 ◽  
Author(s):  
Stéphane C Berneau ◽  
Peter T Ruane ◽  
Daniel R Brison ◽  
Susan J Kimber ◽  
Melissa Westwood ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Embryo Implantation ◽  
Giant Cells ◽  
Ishikawa Cell ◽  
Altered Expression ◽  
Ishikawa Cells ◽  
Cell Layers ◽  
Endometrial Epithelium ◽  
Vitro Model

At the onset of pregnancy, embryo implantation is initiated by interactions between the endometrial epithelium and the outer trophectoderm cells of the blastocyst. Osteopontin (OPN) is expressed in the endometrium and is implicated in attachment and signalling roles at the embryo–epithelium interface. We have characterised OPN in the human endometrial epithelial Ishikawa cell line using three different monoclonal antibodies, revealing at least nine distinct molecular weight forms and a novel secretory pathway localisation in the apical domain induced by cell organisation into a confluent epithelial layer. Mouse blastocysts co-cultured with Ishikawa cell layers served to model embryo apposition, attachment and initial invasion at implantation. Exogenous OPN attenuated initial, weak embryo attachment to Ishikawa cells but did not affect the attainment of stable attachment. Notably, exogenous OPN inhibited embryonic invasion of the underlying cell layer, and this corresponded with altered expression of transcription factors associated with differentiation from trophectoderm (Gata2) to invasive trophoblast giant cells (Hand1). These data demonstrate the complexity of endometrial OPN forms and suggest that OPN regulates embryonic invasion at implantation by signalling to the trophectoderm.

scholarly journals Osmotic stress induces JNK-dependent embryo invasion in a model of implantation

Reproduction ◽  
2018 ◽  
Author(s):  
Peter T Ruane ◽  
Rebekka Koeck ◽  
Stephane C Berneau ◽  
Susan J Kimber ◽  
Melissa Westwood ◽  
...  
Keyword(s):  
Acute Stress ◽  
Giant Cells ◽  
Blastocyst Stage ◽  
Dependent Manner ◽  
Success Rates ◽  
Ishikawa Cell ◽  
Endometrial Cells ◽  
Epithelial Cell Layer ◽  
Endometrial Epithelium

In vitro culture during assisted reproduction technologies (ART) exposes pre-implantation embryos to environmental stressors, such as non-physiological nutritional, oxidative and osmotic conditions. The effects on subsequent implantation are not well understood but could contribute to poor ART efficiency and outcomes. We have used exposure to hyperosmolarity to investigate the effects of stress on the ability of embryos to interact with endometrial cells in an in vitro model. Culturing mouse blastocysts for 2h in medium with osmolarity raised by 400mOsm induced blastocoel collapse and re-expansion, but did not affect subsequent attachment to, or invasion of, the endometrial epithelial Ishikawa cell line. Inhibition of stress-responsive c-Jun N-terminal kinase (JNK) activity with SP600125 did not affect the intercellular interactions between these embryos and the epithelial cells. Four successive cycles of hyperosmotic stress at E5.5 had no effect on attachment, but promoted embryonic breaching of the epithelial cell layer by trophoblast giant cells in a JNK-dependent manner. These findings suggest that acute stress at the blastocyst stage may promote trophoblast breaching of the endometrial epithelium at implantation, and implicates stress signalling through JNK in the process of trophectoderm differentiation into the invasive trophoblast necessary for the establishment of pregnancy. The data may lead to increased understanding of factors governing ART success rates and safety.

scholarly journals Trophoblast attachment to the endometrial epithelium elicits compartment-specific transcriptional waves in an in-vitro model

2020 ◽  
Author(s):  
Paula Vergaro ◽  
Gustavo Tiscornia ◽  
Filippo Zambelli ◽  
Amelia Rodríguez ◽  
Josep Santaló ◽  
...  
Keyword(s):  
In Vitro Model ◽  
Endometrial Epithelium ◽  
Vitro Model

scholarly journals Endometrial pyruvate kinase M2 is essential for decidualization during early pregnancy

2020 ◽  
Vol 245 (3) ◽  
pp. 357-368 ◽  
Author(s):  
Yan Su ◽  
Sujuan Guo ◽  
Chunyan Liu ◽  
Na Li ◽  
Shuang Zhang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Pyruvate Kinase ◽  
Early Pregnancy ◽  
Stromal Cells ◽  
Embryo Implantation ◽  
Pyruvate Kinase M2 ◽  
Ishikawa Cells ◽  
Implantation Sites

Embryo implantation is essential for normal pregnancy. Decidualization is known to facilitate embryo implantation and maintain pregnancy. Uterine stromal cells undergo transformation into decidual cells after embryo attachment to the endometrium. Pyruvate kinase M2 (PKM2) is a rate limiting enzyme in the glycolysis process which catalyzes phosphoenolpyruvic acid into pyruvate. However, little is known regarding the role of PKM2 during endometrial decidualization. In this study, PKM2 was found to be mainly located in the uterine glandular epithelium and luminal epithelium on day 1 and day 4 of pregnancy and strongly expressed in the decidual zone after embryo implantation. PKM2 was dramatically increased with the onset of decidualization. Upon further exploration, PKM2 was found to be more highly expressed at the implantation sites than at the inter-implantation sites on days 5 to 7 of pregnancy. PKM2 expression was also significantly increased after artificial decidualization both in vivo and in vitro. After PKM2 expression was knocked down by siRNA, the number of embryo implantation sites in mice on day 7 of pregnancy was significantly reduced, and the decidualization markers BMP2 and Hoxa10 were also obviously downregulated in vivo and in vitro. Downregulated PKM2 could also compromise cell proliferation in primary endometrial stromal cells and in Ishikawa cells. The migration rate of Ishikawa cells was also obviously suppressed by si-PKM2 according to the wound healing assay. In conclusion, PKM2 might play an important role in decidualization during early pregnancy, and cell proliferation might be one pathway for PKM2 regulated decidualization.

scholarly journals The effects of hyaluronate-containing medium on human embryo attachment to endometrial epithelial cells in vitro

2020 ◽  
Vol 2020 (2) ◽  
Author(s):  
Peter T Ruane ◽  
Chelsea J Buck ◽  
Phoebe A Babbington ◽  
Wedad Aboussahoud ◽  
Stéphane C Berneau ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Embryo Transfer ◽  
Human Embryo ◽  
Research Network ◽  
Human Embryos ◽  
Ishikawa Cell ◽  
Practitioner Training ◽  
Cell Layers ◽  
Endometrial Epithelial Cells

Abstract STUDY QUESTION Does embryo transfer medium containing hyaluronate (HA) promote the attachment phase of human embryo implantation? SUMMARY ANSWER HA-containing medium does not promote human blastocyst attachment to endometrial epithelial cells in vitro. WHAT IS KNOWN ALREADY Embryo transfer media containing high concentrations of HA are being used to increase implantation and live birth rates in IVF treatment, although the mechanism of action is unknown. STUDY DESIGN, SIZE, DURATION Expression of HA-interacting genes in frozen-thawed oocytes/embryos was assessed by microarray analysis (n = 21). Fresh and frozen human blastocysts (n = 98) were co-cultured with human endometrial epithelial Ishikawa cell layers. Blastocyst attachment and the effects of a widely used HA-containing medium were measured. PARTICIPANTS/MATERIALS, SETTING, METHODS Human embryos surplus to treatment requirements were donated with informed consent from several ART centres. Blastocyst-stage embryos were transferred at day 6 to confluent Ishikawa cell layers; some blastocysts were artificially hatched. Blastocyst attachment was monitored from 1 to 48 h, and the effects of blastocyst pre-treatment for 10 min with HA-containing medium were determined. MAIN RESULTS AND THE ROLE OF CHANCE Human embryos expressed the HA receptor genes CD44 and HMMR, hyaluronan synthase genes HAS1–3, and hyaluronidase genes HYAL1–3, at all stages of preimplantation development. Attachment of partially hatched blastocysts to Ishikawa cells at 24 and 48 h was related to trophectoderm grade (P = 0.0004 and 0.007, respectively, n = 34). Blastocysts of varying clinical grades that had been artificially hatched were all attached within 48 h (n = 21). Treatment of artificially hatched blastocysts with HA-containing medium did not significantly affect attachment at early (1–6 h) or late (24 and 48 h) time points, compared with control blastocysts (n = 43). LIMITATIONS, REASONS FOR CAUTION Using an adenocarcinoma-derived cell line to model embryo-endometrium attachment may not fully recapitulate in vivo interactions. The high levels of blastocyst attachment seen with this in vitro model may limit the sensitivity with which the effects of HA can be observed. WIDER IMPLICATIONS OF THE FINDINGS Morphological trophectoderm grade can be correlated with blastocyst attachment in vitro. HA-containing medium may increase pregnancy rates by mechanisms other than promoting blastocyst attachment to endometrium. STUDY FUNDING/COMPETING INTEREST(S) This work was funded by a grant from the Wellbeing of Women, the NIHR Local Comprehensive Research Network and NIHR Manchester Clinical Research Facility, the Department of Health Scientist Practitioner Training Scheme, and the Ministry of Higher Education, The State of Libya. None of the authors has any conflict of interest to declare.

scholarly journals An in vitro Model for Blood Brain Barrier Permeation

2002 ◽  
Vol 70 (4) ◽  
pp. 317-322 ◽  
Author(s):  
Bauer R. ◽  
Lauer R. ◽  
Linz B. ◽  
Pittner F. ◽  
Peschek G.A. ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
Phosphodiesterase Inhibitor ◽  
Culture Conditions ◽  
Brain Barrier ◽  
Type Iv ◽  
Blood Brain ◽  
Cell Layers ◽  
Vitro Model ◽  
Brain Microvessel

The ability to permeate accross the blood brain barrier (BBB) is essential for drugs acting on the central nervous system (CNS). Thus, systems that allow rapid and inexpensive screening of the BBB-permeability properties of novel lead compounds are of great importance for speeding up the drug discovery process in the CNS-area. We used immortalized porcine brain microvessel endothelial cells (PBMECICl-2) to develop a model for measurement of blood-brain barrier permeation of CNS active drugs. Investigation of different cell culture conditions showed, that a system using C6 astrocyte glioma conditioned medium and addition of a cyclic AMP analog in combination with a type IV phosphodiesterase inhibitor (R020-1724) leads to cell layers with transendothelial electrical resistance values up to 300 Ω.cm2. Permeability studies with U-[14C]sucroseg ave a permeability coefficient Pe of 3.24 + 0.14 × 10−4 cm/min, which is in good agreement to published values and thus indicates the formation of tight junctions in vitro.

ABSTRACTS: 3
Development of an in vitro model for human embryo implantation and progesterone regulation

2008 ◽  
Vol 60 (1) ◽  
pp. 86-86
Author(s):  
Sujata Lalitkumar ◽  
PGL Lalitkumar ◽  
ChunXia Meng ◽  
Kristina Gemzell-Danielsson
Keyword(s):  
Human Embryo ◽  
In Vitro Model ◽  
Embryo Implantation ◽  
Vitro Model

scholarly journals Dysregulations of Expression of Genes of the Ubiquitin/SUMO Pathways in an In Vitro Model of Amyotrophic Lateral Sclerosis Combining Oxidative Stress and SOD1 Gene Mutation

2021 ◽  
Vol 22 (4) ◽  
pp. 1796
Author(s):  
Audrey Dangoumau ◽  
Sylviane Marouillat ◽  
Roxane Coelho ◽  
François Wurmser ◽  
Céline Brulard ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Amyotrophic Lateral Sclerosis ◽  
Motor Neurons ◽  
Genetic Alterations ◽  
Sod1 Gene ◽  
Altered Expression ◽  
Expression Of Genes ◽  
Vitro Model ◽  
Lateral Sclerosis

Protein aggregates in affected motor neurons are a hallmark of amyotrophic lateral sclerosis (ALS), but the molecular pathways leading to their formation remain incompletely understood. Oxidative stress associated with age, the major risk factor in ALS, contributes to this neurodegeneration in ALS. We show that several genes coding for enzymes of the ubiquitin and small ubiquitin-related modifier (SUMO) pathways exhibit altered expression in motor neuronal cells exposed to oxidative stress, such as the CCNF gene mutated in ALS patients. Eleven of these genes were further studied in conditions combining oxidative stress and the expression of an ALS related mutant of the superoxide dismutase 1 (SOD1) gene. We observed a combined effect of these two environmental and genetic factors on the expression of genes, such as Uhrf2, Rbx1, Kdm2b, Ube2d2, Xaf1, and Senp1. Overall, we identified dysregulations in the expression of enzymes of the ubiquitin and SUMO pathways that may be of interest to better understand the pathophysiology of ALS and to protect motor neurons from oxidative stress and genetic alterations.

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