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

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.


Results
K252a interferes with in vitro cell reassembly. We first determined whether protein kinase inhibitor K252a influenced cell viability and attachment in the first 48 hours following cell seeding. The proportion of all viable cells amongst K252a concentration range did not differ when compared to that of no treatment control (Fig. 1a). Similarly, the proportion of attached live cells was comparable to that of no treatment control (Fig. 1b). This indicates, that K252a did not affect cell viability during the experiments and suggests that its presence did not disturb cell attachment which had already occurred 48 hours after seeding.
We next analysed morphologically the effects of K252a on cellular reaggregation in our in vitro system. Initially, we validated the structured reaggregation of Sertoli and peritubular cells in cord-like structures by immunohistochemical analyses of control cultures, not treated -with K252a (Fig. 2). We confirmed that coalescing round aggregates were connected by alpha-smooth muscle actin (αSMA) positive peritubular cells, and a preceding formation of elongated cord-like structures within a week of in vitro culture ( Fig. 2A). Further analysis of cord-like structures cross-sections revealed their spatial cytoarchitecture -Sertoli cells were located centrally  www.nature.com/scientificreports www.nature.com/scientificreports/ to 11.9% (aggregate size) and 2.6 to 10% (aggregate number), while for vehicle control the values ranged from 1.3 to 15.9% and from 0.8 to 14.6%, respectively. These CV values confirmed that the culture approach could be performed with high reproducibility and reliability. Subsequently, we compared aggregate number and size in treatment groups to vehicle control (Fig. 3B,C). While the number of aggregates was similar for all K252a concentrations (Fig. 3B), treatment with K252a in the range of 200 nM-5 µM significantly decreased median aggregate size (Fig. 3C). The IC50 for the inhibitory effect of K252a on median aggregate size was 203.3 ± 47.8 nM (Fig. 3D).
K252a prevents cell reassembly in large aggregates and those effects are irreversible. Since we observed a difference in aggregate size between treatment groups and vehicle control, we evaluated in further detail the effect of K252a on cell reassembly by defining a range of aggregate size categories ( Supplementary  Fig. 1). We then quantified the aggregate numbers from each size category for treatment groups and vehicle control (Fig. 4). Treatment with 200 nM-5 µM K252a significantly reduced formation of large aggregates (>6000 µm 2 ; Fig. 4e-h), while aggregate number in the smallest size category increased under exposure to 2 µM and 500 nM K252a (Fig. 4f,g).
We also determined whether initial short-term exposure to K252a followed by compound wash-out would rescue cellular reaggregation ( Supplementary Fig. 2). Withdrawal experiments confirmed that decrease in the number of large aggregates was irreversible when cells were exposed for 2 days to 2 µM and 500 nM K252a ( Supplementary Fig. 2c,d). However, inhibitory effects of 200 nM K252a on aggregate numbers were rescued after withdrawal ( Supplementary Fig. 2b).  In vitro cord-like structure formation is persistently disturbed by constant exposure to K252a. Since large aggregate numbers were significantly decreased following constant exposure to 200 nM-5 µM K252a (Fig. 3C), we performed a two-week follow up on the effect of high K252a concentrations (5 µM and 500 nM) on aggregate morphological changes (Fig. 5). Aggregates in vehicle control had already interconnected via bundles of elongated cells and initiated coalescence at day 7. These morphological changes progressed further towards the formation of larger and compact aggregates by day 14 (Fig. 5a,d), while exposure to 5 µM K252a completely inhibited aggregate connections (Fig. 5c,f). Treatment with 500 nM K252a affected aggregate interconnection such that only single dispersed elongated cells extended from the aggregates and did not form further dense bundles as in vehicle controls (Fig. 5b,e).

Discussion
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. We validated the model as a quantitative assay with robust endpoints that enable analysis of in vitro cellular reaggregation and cord-like structure formation patterns.
Our results reveal a dose-dependent disturbance of cell reassembly and a shift from larger to smaller aggregates at IC50 of 203.3 nM for K252a. Concordant to our data, a study using a cell-based assay with immature rat testicular cells demonstrated disruption of cord formation by characterising inhibition of reaggregation with parameters similar to ours (i.e. aggregate diameter and distance between aggregates) 19 . However, in contrast to complete inhibition of aggregate formation observed at 5 nM K252a in Gassei et al. 19 , testicular cells in our system reassembled, albeit in significantly smaller aggregates, across the entire K252a concentration range applied. Other studies exposing entire undifferentiated rat gonadal primordia to 100 nM K252a described a complete inhibition of testis cord formation 18,21 . It can be argued that this discrepancy is due to that those studies used entire organs, where tissue integrity and cellular associations are preserved. Another in vitro study using second trimester human foetal testes showed altered number and proliferation of testicular somatic cells, i.e. Sertoli and peritubular cells, when gonads were treated with 100 nM K252a for 48 hours 20 . It is commonly acknowledged that www.nature.com/scientificreports www.nature.com/scientificreports/ adult testicular somatic cells are non-proliferative 22 . Although we have not assessed cell proliferation in our study, we did not observe change in cell viability after exposure to K252a for 48 hours. Nevertheless, it is known that progression through different stages of foetal and postnatal testis tubulogenesis is marked by changes in proliferation and protein expression in somatic testicular cells [23][24][25] .
It is also plausible that there are species-and age-related differences in specific signalling pathways between human and rodents. As an example, studies addressing protein and gene expression of neurotrophins in testis have shown species-and cell type-specific expression of those factors and the expression was also age-dependent 18,[26][27][28][29] . It was assumed that neurotrophins and their receptors are involved in seminiferous tubulogenesis and some of these studies used K252a at 100 nM to functionally challenge cord formation 18,20,21 . However, apart from neurotrophin receptor tyrosine kinases, K252a targets several protein kinases in the IC50 range between 3 and 25 nM 17,30,31 . Given this general ability to interfere with tubulogenesis at various levels and targets, we did choose this broad spectrum inhibitor as it was perfectly suited to test our system and to identify valid parameters that reflect the impact of this pharmacological challenge. However, applying an array of selective inhibitors at a range of concentrations to define dose-response curves will be required to dissect cellular mechanisms of testis tubulogenesis 32 . Furthermore, since no IC50 values were calculated in the aforementioned studies the conclusions that can be drawn may be limited 18,21,33 .
Tissue or organ culture approaches enable investigation of cell signalling pathways of seminiferous tubulogenesis [34][35][36] . However they are limited by low-throughput and do not allow simultaneous exploration of different conditions. Here, we validated the application of single cell suspensions to explore in vitro testicular cord formation. We propose that our cell-based assay with quantitative endpoints relevant to cell aggregation can facilitate future studies that address stage-timed proliferation, gene and protein molecular analyses for specific cellular mechanisms and processes. To characterise the role of cellular migration and contractility during cord morphogenesis 1,10 , this cell culture system can -in more specific applications -be coupled with advanced analytical techniques such as traction force microscopy [37][38][39] . It may then be extended to assess the effects and mechanisms of pharmaceutical toxicants and chemotherapy drugs on testis cell types, which are currently tested predominantly on rodent models [40][41][42] .
In conclusion, we demonstrate the responsiveness of primary human testicular cells to experimental perturbations by exogenous administration of a protein kinase inhibitor known to interfere with in vitro cord formation. We further propose our in vitro model as a functional platform to quantitatively investigate the processes of seminiferous tubulogenesis by pharmacological interference.

Materials and Methods
testis tissue source and isolation of testicular cell suspensions. In total, 22 testes were procured from 22 adult gender dysphoria patients undergoing sex confirming surgery at the Urological Department of University Clinic of Essen, Germany. Ethical approval for the use of testicular tissue was obtained from the ethical committee of the Ärztekammer Westfalen-Lippe (no. 2012-555-f-S). Prior to surgery, patients had undergone gender confirming hormone therapy with anti-androgens (cyproterone acetate) and oestrogens. Clinical standard procedure was to discontinue cross-sex hormone therapy two weeks before surgery. We have previously published a detailed characterisation of our patient cohort regarding steroidogenesis status, testicular tissue histology and evaluation of degree of spermatogenesis via histological and flow cytometry analyses 16,[43][44][45][46] . All experiments were performed in accordance with the relevant institutional guidelines and regulations. Testes were used after the patients provided written informed consent. Testes were transported in chilled Dulbecco's modified Eagle's medium low Glucose (1 g/l) + Pyruvate + L-Glutamine (31885; Gibco Life Technologies, USA) (DMEM) at 4 °C. Testes were decapsulated and 16 fragments of approx. 100 mg were dissected mechanically into 1-3 mm 3 fragments with scissors. To obtain testicular cell suspensions, each sample was subjected individually to a two-step enzymatic digestion following a previously described protocol 16 . Each cell suspension that was generated from the 22 testes was considered an independent biological experiment. cell culture conditions. Enzymatically generated testicular cell suspensions were seeded at density of 5×10 5 cells/well in 24-well plates (83.3922; Sarstedt AG, Nümbrecht, Germany). Cells were cultured in DMEM, supplemented with 1% (v/v) MEM non-essential amino acids (11140; Gibco Invitrogen, Darmstadt, Germany), 1% (v/v) Anti/Anti (110X15240-062; Gibco Life Technologies, Grand Island, NY, USA). The medium for cultured cells analysed by downstream immunohistochemical analysis was additionally supplemented with 10% (v/v) KnockOut TM serum replacement (10828010; Gibco, Life Technologies, USA). The commercially available protein kinase inhibitor K252a (K1639; Sigma, Steinheim, Germany), isolated from Nonomuraea longicatena, was applied at the time of cell plating. The compound was dissolved in dimethyl sulfoxide, DMSO (D5879; Sigma-Aldrich, Steinheim, Germany). Intermediate stocks of 10 µM, 4 µM, 2 µM, 1 µM, 0.1 µM and 0.01 µM were prepared by diluting the stock solution in culture medium. The final concentration range for K252a treatment groups that were attained in the wells containing plated testicular cells were 5 µM, 2 µM, 500 nM, 200 nM, 100 nM, 10 nM, 5 nM and 1 nM. For control experiments, cells were either cultured in culture medium alone (no treatment control) or DMSO was added to culture medium (vehicle control) in appropriate dilution (0.5% v/v) comparable to that in treatment groups. Cells were incubated at 35 °C and 5% CO 2 . Upon medium renewal 48 h after initial cell seeding 16 , the medium contained corresponding concentrations of K252a (1 nM-5 µM). Cells were then cultured for another 1-3 days before image acquisition for quantitative analysis. The experimental design was based on previously published studies exploring the effects of K252a within a 48-72-hour window of challenge [18][19][20][21]30 .
Additional cultures were exposed to vehicle control, 500 nM and 5 µM K252a for two weeks without medium renewal to account for the influence of high K252a concentrations on cellular re-aggregation over extended culture periods under constant exposure. Images were acquired on days 7 (n = 13) and 14 (n = 11) for qualitative analysis.
In a second set of experiments, K252a was introduced at cell seeding at a defined range of concentrations (100 nM-2 µM) for 48 h after seeding (withdrawal experiments) and was then replaced by culture medium only, which did not contain any K252a or DMSO. Cells were cultured for another 2-3 days when image acquisition for quantitative analysis was performed. evaluation of cell viability. Testicular cells were cultured for 48 h under control conditions (no treatment or vehicle) and under exposure to 1 nM, 100 nM and 5 µM K252a. Cell viability was determined after 48 hours of single K252a challenge as this was the first time point when medium exchange was performed following our published protocol 16 . Medium containing the floating cell population was aspirated, pooled from the triplicates and pelleted at 536 g for 7 minutes. The pellet was resuspended in DMEM and live vs. dead cell numbers were determined via viability assessment with 0.4% (w/v) trypan blue. To enumerate live and dead cells in the adherent cell population, adherent cells were detached by incubation with 0.05% trypsin/EDTA for 1 minute at room temperature. Reaction was stopped by adding equal volume of DMEM to each well. Proportion of all live cells (in floating and adherent fraction) expressed out of total number of live and dead cells (in floating and attached fraction) was calculated. Accordingly, percentage live cells attached, expressed as the proportion of live cells in the attached fraction out of the total number of live cells (in floating and attached cell fraction) was evaluated. Each condition was performed in technical triplicates and in at least five independent experiments per condition. immunohistochemical staining of cross-sections and whole cord-like structures formed in vitro. For detection of marker proteins of Sertoli and peritubular cells, a double staining for SRY-related high mobility box 9 (SOX9) and alpha-smooth muscle actin (αSMA) was performed on fixed aggregates whole and cross-sections of paraffin embedded cord-like structures 47 . The following antibodies were used: polyclonal rabbit anti-SOX9 (Dilution 1:100; AB5535 Millipore), monoclonal mouse anti-αSMA (Dilution 1:500 or 1:1000; A2547; Sigma-Aldrich, Germany). SOX9 antibody was visualized by secondary horseradish peroxidase-labelled chicken anti-rabbit IgG (Dilution 1:100; SC-2955, Santa Cruz Biotechnology, Paso Robles, CA, USA). Diaminobenzidine (DAB), used as a substrate, generated a brown signal at immunopositive sites. Alpha-SMA was visualized by secondary alkaline phosphatase-labelled chicken anti-mouse IgG (Dilution 1:100; SC-2958, Santa Cruz Biotechnology, Paso Robles, CA, USA). The use of DAKO REAL detection system alkaline phosphatase (K5005; DAKO, Denmark) produced a red precipitate in immunopositive regions. For SOX9 and αSMA staining, a previously described protocol was used 16 . Briefly, whole aggregates and aggregate cross-sections were washed twice with distilled water before blocking of non-specific binding by incubation with 5% chicken serum diluted in Scientific RepoRtS | (2020) 10:8935 | https://doi.org/10.1038/s41598-020-65924-y www.nature.com/scientificreports www.nature.com/scientificreports/ 0.1% bovine serum albumin, BSA. Samples were incubated overnight at 4 °C with a mix of SOX9 and αSMA antibodies, both diluted in 0.1% BSA. Incubation with secondary antibodies was performed on the next day after several washing steps, followed by further washing steps before development of colour reaction with DAB for 4-6 minutes. Afterwards, DAKO REAL detection system for 2-3 minutes was applied, interrupted by washing steps with Tris-buffered saline (TBS) in between. Samples were counterstained with haematoxylin (Mayer's hemalaum solution; 1092490500; Merck Millipore, Darmstadt, Germany) and mounted in Faramount (S3025; DAKO, Glostrup, Denmark).

Micrograph acquisition and time
image processing and digital data generation. All 48 micrographs for each condition within an experiment were processed with Fiji software 49 . A sequence of macro instructions was created for automatic quantitative analysis. After a flat field correction, thresholding was used to generate a binary mask of the aggregate boarders. This mask was further processed to smooth the outline of the aggregates and fill small holes without changing the overall size (Fig. 6a,b). These aggregates were analysed with the "analyse particle" routine in Fiji.
The intra-assay coefficient of variation, CV, (n = 5) was used as a measure of the validity of the data acquisition approach and the macro instructions created in Fiji. It also served to confirm the reproducibility of the established culture approach. A mean of individual aggregate size was calculated for each of the 16 images taken per replicate and the total number of aggregates per image was recorded. Then, a mean value was calculated out of the 16 summary values. These three values were used to calculate the intra-assay CV for each individual experiment.
Data about aggregate number and size from treatment conditions and controls were subjected to the same processing algorithm as for intra-assay CV. Arithmetic means calculated from the three values for controls and treatment conditions were used for statistical analysis. To calculate IC50 for the parameter aggregate size, the aggregate size for each set of experiments was normalized to the aggregate size of the vehicle control. The mean relative aggregate sizes were plotted against the K252a concentration and fitted with a logistic function using Origin (OrginLab 2019).