Chemicals and reagents

DBP (CAS No 84-74-2, purity > 99%), dimethyl sulfoxide (DMSO), paraformaldehyde, 4´,6-diamidino-2-phenylindole dihydrochloride (DAPI), collagenase, hyaluronidase, DNase, Triton X-100, 3-(4,5-dimethyl2-yl)-2,5-diphenyltetrazolium bromide (MTT), nonessential amino acids, epidermal growth factor (EGF), sodium pyruvate, sodium lactate and bovine serum albumin (BSA) were purchased from Sigma Aldrich (St. Louis, MO, USA). Phosphate-Buffered Saline (PBS) and Minimum Essential Medium (MEM no glutamine, no phenol red) were purchased from Gibco (Thermo Fischer Scientific, NY, USA). Tween 20 and NaCl were purchased from Fisher (Loughborough, UK). Penicillin–streptomycin (P/S), trypsin solution (0.05%) and trypsinedta were obtained from Gibco (Life Technologies, Paisley, UK). Insulin Transferrin-Selenium (ITS) premix was purchased from Corning Discovery Labware, Inc. (Bedford, MA, USA). Matrigel Basement Membrane Matrix was obtained from Corning (MA, USA). Anti-SOX9 monoclonal (ab207677), anti-ZO-1 monoclonal (ab221547), anti-N-cadherin monoclonal (ab98952), anti-DAZL monoclonal (ab215718) and normal goat serum (blocking agent, ab7481) were obtained from Abcam (Cambridge, UK). Anti-CYP11A1 (PA5-109,610), Anti-StAR (PA5-21,687), Alexa Fluor 568 Phalloidin (A12380), Goat anti-rabbit IgG Alexa Fluor 488 (A-11008), Goat anti-mouse IgG Alexa Fluor 647 (A-21235), HSD17B3 polyclonal antibody (PA5-30,063), CYP17A1 monoclonal antibody (MA5-35,632), and LHR polyclonal antibody (PA5-97,923) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

3D testicular co-culture and DBP exposure

Pregnant C57Bl/6NCrl mice at 8 weeks of age were purchased from (Charles River, Sulzfeld, Germany) and housed in Makrolon cages in a room controlled for temperature and humidity at the Experimental Core Facility (ECF) at Stockholm University. Tap water and food pellets were provided ad libitum. After birth, male pups at the age of 7-day postnatal (PND 7) were euthanized by decapitation, and the testes were collected. The seminiferous tubules were enzymatically dissociated and dispersed into a single cell suspension using a two-step digestion protocol described by Wegner and colleagues with modifications (Wegner et al. 2013). In brief, to harvest the undescended testes from pups, pressure was applied on each side of the lower abdomen to force testicular descent. A small lateral abdominal incision was made using sterile scissors to remove the testes. The testes were transferred to a cell culture dish containing enough fresh MEM to submerge testes. Next, the testes were decapsulated with sterile fine forceps under a dissecting microscope to tear the tunica albuginea and squeeze the seminiferous tubules out. Once isolated, the aggregated seminiferous tubules were transferred into a new dish of MEM and mechanically desegregated and cut into four to six pieces to give enzymes greater access to tissue. The tissues were washed with MEM and allowed to settle at the bottom of a 15 ml Falcon tube to remove excess blood, which may interfere with the digestive enzymes.

In the first digestion step, the testicular tissues were digested using a mixture of collagenase (1 mg/ml), DNase I (0.001 mg/ml), and hyaluronidase (1 mg/ml) in MEM (enzyme cocktail A) for 20 min at 37 °C. The tube was gently agitated at the midpoint of incubation. The supernatant was removed and the tissues were washed with 10 mL of ice-cold MEM for 10 min. In the second-digestion step, MEM was gently removed and the remaining small tissues were processed in another culture medium supplemented with a mixture of collagenase (1 mg/ml) and DNase I (0.001 mg/ml) (enzyme cocktail B) for 20 min at 37 °C. Subsequently, the enzyme cocktail B was removed and the tissues were incubated in ice-cold MEM for 10 min before reapplying cocktail B for additional 20 min at 37 °C. The supernatant was then removed and the tissues were washed with MEM on ice for 10 min, followed by incubation with 0.05% trypsin EDTA (a volume of 5 × the amount of testicular tissue) for 3 min at 37 °C. Next, the trypsin inhibitor solution, consisting of 0.4% DNase I in soybean trypsin inhibitor 1X, was added to the pellets and centrifuged at 150 g for 5 min. The supernatant was removed and the pellets were resuspended in fresh trypsin inhibitor solution followed by a second round of centrifugation at 150 g for 5 min. Finally, the cell pellet was resuspended in trypsin inhibitor solution before being dispersed with a sterile Pasteur pipette and filtered through a nylon mesh cell strainer.

The dissociated testicular cells were resuspended in serum-free MEM containing nonessential amino acids (at a final concentration of 0.001 mM), sodium pyruvate (0.01 mM), sodium lactate (0.8 µM), EGF (0.003 µg/ml), penicillin–streptomycin (1% vol/vol) and 1% ITS culture supplement. The viability and the count of the isolated cells were assessed by hemocytomer. After that, cells were seeded in Primaria fibronectin-coated plates (96-well plate or 6-well plate). To prepare fibronectin-coated plates, stock fibronectin solution was diluted in Milli-Q water (1:100) and incubated for 30 min at 4 °C. The fibronectin solution was added to the plates and incubated for at least 2 h at 37 °C. The plates were washed and stored in PBS until ready for cell seeding. For the 96-well plates, the cells were seeded at a density of 4 × 104 cells/well in 100 µl culture media followed by the addition of 15 μl ice-cold Matrigel extracellular matrix overlay (at a final concentration of 150 µg/ml) to the center of each well and gently swirled with cell suspension to facilitate in vivo-like three-dimensional scaffold. For the 6-well plates, the concentration of the cells was 2 × 106 cells/well in 1 ml media. After a 48-h acclimation period, the media in the 3D testicular co-cultures were replaced with new media containing DBP at a range of concentrations, 1 nM, 10 nM, 100 nM, 500 nM, 1 µM, 5 µM, 10 µM, 50 µM and 100 µM for 7 days. Controls were exposed to 0.1% DMSO only. The exposure media were replaced every other day. The culture media were collected at the end of the experiment and stored at  – 80 °C for subsequent ELISA testosterone measurement. A schematic representation of the testicular co-culture workflow is illustrated in Fig. 1.

Fig. 1

Schematic illustration of the 3D testicular co-culture workflow for mechanistic screening of reproductive toxicants. Di-n-butyl phthalate (DBP) was used as a model compound to illustrate the utility of this in vitro model. Figure created with BioRender.com

The animal procedures were approved by the Regional Animal Ethical Committee in accordance with the Swedish legislation on Animal Experimentation (Animal welfare act SFS1998:56) and the European Union Directive on the Protection of Animals Used for Scientific Purposes (2010/63/EU).

Cell viability analysis

After the DBP treatment, the cell viability was measured by the MTT assay. The culture media was replaced with 100 µl/well of 0.5 mg/ml MTT reagent dissolved in media. After 3 h incubation at 37 °C, the MTT solution was removed, and the generated formazan crystals were solubilized by adding 50 µL DMSO to each well. The absorbance of formazan was measured at 590 nm using a SpectraMax i3 microplate reader (Molecular Devices, San Jose, CA, USA).

Testosterone analysis

Testosterone levels were measured in the collected cell culture media using a colorimetric competitive enzyme-linked immunosorbent ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA) in accordance with the manufacturer’s instructions. The absorbance was measured at 405 nm using a SpectraMax i3 microplate reader (Molecular Devices, San Jose, CA, USA).

Immunofluorescence staining of cell-specific markers and key testicular proteins

To analyze protein levels by immunofluorescence and high-content imaging, the co-cultures were washed with PBS and fixed with 4% paraformaldehyde for 20 min. For permeabilization, the cells were incubated with 0.25% Triton X-100 for 10 min followed by three times washing with PBS for 5 min. The cells were then blocked for 3 h in blocking buffer containing 10% normal goat serum, 1% BSA and 0.05% Tween 20 diluted in PBS. Primary antibodies against key testicular proteins were incubated in 1% BSA diluted in PBS overnight at 4 °C. Cells were washed three times in PBS and then incubated with secondary antibodies conjugated with 488, 555 or 647 fluorophores diluted in PBS for 1 h at room temperature. DAPI (0.5 µg/ml) was used for nuclear counterstaining. The total cell count was determined by DAPI staining of all nuclei. The analysis images were acquired using a 10 × air objective on ImageXpress micro XLS confocal High-content analysis system (Molecular devices, Sunnyvale, CA, USA). Nine sites per well were analyzed with MetaXpress Software (Molecular devices, Sunnyvale, CA, USA) after digital acquisition using the cell scoring module. Representative images for the figures were taken using 20 × water immersion objective.

To evaluate the impact of DBP on the main testicular cell populations, the cells were stained with cell-specific antibodies, including RNA splicing and binding protein (anti-DAZL; germ-cell marker), cholesterol side-chain cleavage enzyme (anti-CYP11A1; Leydig cell marker) and transcription factor SOX9 (anti-SOX9; Sertoli cells). The cells were co-stained for SOX9 and CYP11A1. The cells were also stained for the steroidogenic acute regulatory protein (StAR). To investigate the effects of DBP on the expression pattern of junctional proteins in Sertoli cells, the 3D testicular co-culture was triple-immunostained for Cadherin-2 (N-Cadherin), Zonula occludens protein 1 (ZO-1) and F-Actin. The fluorescence intensity was quantified using the cell scoring module, integrated fluorescence application mode via MetaXpress high-content image acquisition and analysis software. The number of cell-marker positive cells divided by DAPI total cells was also analyzed to determine the DBP effect on the relative number of each cell type.

Western blot analysis of steroidogenic proteins

To analyze protein levels by western blot cells were washed with PBS and trypsinized, and the detached cells were collected and lysed with RIPA buffer (150 mM NaCl, 0.1% Triton X-100, 0.5% Sodium Deoxycholate, 0.1% SDS, 50 mM Tris–HCl, 1% protease inhibitors, pH 8). The protein concentration was determined by Lowry assay. Western blot was performed as previously described (Källsten et al. 2022b). Primary antibodies were diluted at 1:1000 and incubated overnight. The blots were subsequently incubated for 1 h with peroxidase conjugated anti-mouse or anti-rabbit diluted at 1:5000. The blot was developed with a chemiluminescence ECL kit with a charge-coupled device (CCD) imager (iBright CL750 Imaging System, ThermoFisher, Rockford, IL, USA), and the optical density was measured with ImageJ software (NIH, USA). The loading control β-actin was used for normalization and the protein levels are shown as percentage of control.

Statistical analysis

All the experimental data are expressed as means ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was applied to analyze differences between the DBP-treated cells and controls. The statistics tests were conducted using GraphPad Prism 8 software (San Diego, CA, USA). The difference is considered to be statistically significant when p-value < 0.05.