Challenging human somatic testicular cell reassembly by protein kinase inhibition \u2013setting up a functional in vitro test system

M Mincheva,C Brenker,J Wistuba,S Schlatt

doi:10.1038/s41598-020-65924-y

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.

Highlights

Signalling pathways and cellular interactions defining initial processes of testis morphogenesis, i.e. cord formation, are poorly understood
In vitro testicular cell culture models of seminiferous tubulogenesis can be applied in a versatile fashion to understand signalling pathways and processes of seminiferous tubulogenesis[10,11,12,13,19]
In this proof of principle study, we demonstrated that our recently described primary human testicular cell culture system[16] is responsive to pharmacological challenge with protein kinase inhibitor K252a with expected inhibitory effects on cord formation

Summary

Introduction

Signalling pathways and cellular interactions defining initial processes of testis morphogenesis, i.e. cord formation, are poorly understood. Rodent embryonic testis culture experiments showed that pre-Sertoli cell aggregation and migration of somatic cells, such as pre-peritubular cells, are relevant steps during cord formation[1,2,5,6] These processes are regulated by paracrine growth factors which act as chemotactic agents that orchestrate cell migration and cell-cell interactions[2,5,6]. An in vitro system resembling these processes might lead to better understanding of the developmental sequences and of causes for testis-related diseases and infertility, many of which originate in early development[7] In this regard, cell suspension-based in vitro culture systems have the advantage over tissue explant-based culture approaches because they facilitate the determination of cellular interactions and pathways regulating testicular tubulogenesis[8]. As a proof of principle, we demonstrate that our model system can be used as a functional assay with quantitative endpoints that could be developed in a tool to interrogate processes of tubulogenesis in future studies

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Conclusion