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Challenging human somatic testicular cell reassembly by protein kinase inhibition -setting up a functional in vitro test system.

Scientific reports (2020-06-04)
M Mincheva, J Wistuba, C Brenker, S Schlatt
ABSTRACT

Signalling pathways and cellular interactions defining initial processes of testis morphogenesis, i.e. cord formation, are poorly understood. In vitro cell-based systems modelling cord formation can be utilised as platforms to interrogate processes of tubulogenesis. We aimed at testing our established cord formation in vitro model using adult human testicular cells as a quantitative assay that can facilitate future studies on cord morphogenesis. We challenged the responsiveness of our system with a broad-spectrum protein kinase inhibitor, K252a. Cultured testicular cells were treated with various K252a concentrations under constant exposure and compound withdrawal. To quantify cell reaggregation changes, we performed computer-assisted phase-contrast image analysis of aggregate size and number. Cell reaggregation was analysed in detail by categorisation of aggregates into size groups and accounting for changes in aggregate number per size category. We found a dose-related disturbance of testicular cell reaggregation. K252a decreased aggregate size (IC50 of 203.3 nM) and reduced the large aggregate numbers. Video recordings revealed that treatment with K252a at a concentration above IC50 interfered with aggregate coalescence into cords. Short-term exposure and compound wash-out induced irreversible decrease in large aggregates. We propose our in vitro model as a functional platform to quantitatively investigate seminiferous tubulogenesis under pharmacological impact.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Streptavidin−Peroxidase from Streptomyces avidinii, lyophilized powder
Sigma-Aldrich
K-252a, from Nonomuraea longicatena, ≥98% (HPLC)
Sigma-Aldrich
Anti-Rabbit IgG (H+L)-Biotin antibody produced in chicken, affinity isolated antibody, lyophilized powder
Sigma-Aldrich
Dimethyl sulfoxide, ReagentPlus®, ≥99.5%
Sigma-Aldrich
Anti-Sox9 Antibody, Chemicon®, from rabbit
Sigma-Aldrich
Monoclonal Anti-Actin, α-Smooth Muscle, clone 1A4, ascites fluid