Mice
Full knockout mice with a germline deletion of Parl (Parltm1.1Bdes (Parl−/−) were provided by Prof. Bart De Strooper (KU Leuven) and generated as described by Cipolat et al.17. Mice were kept in small groups in a temperature- and humidity-controlled room with a 12-h light/dark cycle. Food and water were provided ad libitum. Breeding was performed using heterozygote (Parl+/−) animals. Parl+/+ animals were used as the wild-type (WT) control, and Parl+/− animals were used in some experiments as an additional control. For tissues other than the testes, male and female animals were used. Animal handling was performed in strict accordance with Governmental Directive 2010/63/EU of the European Parliament and of the Council of September 22, 2010 further amended by regulation (EU) 2019/1010, institutional animal care regulations and ARRIVE guidelines.
Western blot, RT-qPCR, ELISA, Complex III and IV activity and ATP detection
Animals at the age of 10 days (P10), 4 weeks (4 W), 6 weeks (6 W), and 8 weeks (8 W) were sacrificed using an overdose of carbon dioxide. Organs (testes/ovaries, brain [separated into brain stem, cerebellum, diencephalon, striatum, and cortex], pituitary gland, spleen, thymus, adrenal glands, and quadriceps) were dissected from the animals and snap-frozen in liquid nitrogen.
Western blotting tissues were homogenized in NP-40 buffer (50 mM Tris pH 7.4 (Merck), 150 mM NaCL, 1% Triton X-100 (Sigma Aldrich) and separated in Nupage 4%–12% Bis-Tris 10 well gels (Invitrogen, Thermo Fisher Scientific) at a protein concentration of 2 µg/µL. Proteins were transferred to Amersham Protran 0.45-µm nitrocellulose blotting membrane (Cytiva). Membranes were blocked for 1.5 h at room temperature (RT) with Roti-Block (Carl Roth) in PBS, incubated overnight at 4°C with the primary antibody in Roti-Block-PBS (for details see Table S1), washed with 0.05% Tween20 in PBS (TPBS), incubated for 1.5 h at RT with the secondary antibody (1:10,000 in Roti-Block-PBS with 0.5% milk, HRP goat anti-rabbit or mouse, Jackson ImmunoResearch), and washed with TPBS again. Amersham ECL Prime Western Blotting detection reagents (Cytiva) and a VersaDoc Imaging System MP 4000 (BioRad) were used to visualize protein bands.
For RNA isolation, a RNeasy Mini Kit (Qiagen) was used according to the instructions using 20 mg (±10%) of testis tissue from 8-week-old mice as the starting sample and including an on-column DNase step with a PureLink DNase Set (Thermo Fisher Scientific). Five hundred nanograms of RNA were reverse-transcribed to cDNA using a Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scientific), as described in the instructions, and included a reverse transcriptase minus control (RT-control). For the RT-qPCR, commercially available TaqMan Gene Expression Assays (Thermo Fisher Scientific) and TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) were used according to the manufacturer’s instructions with 1 µL of cDNA in 10 µL of reaction mix per reaction. The RT-qPCR was performed in triplicate for each sample in each gene using a LightCycler96 (Roche). As a negative control, cDNA was replaced by nuclease-free water for each gene.
The concentration of LH in the pituitary gland was measured using a Mouse LH Beta ELISA Kit (Abcam). A pituitary gland was disrupted and homogenized in 100 µL of the provided 1X Cell Extraction Buffer PTR, the protein concentration was measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), and samples were used at a dilution of 1:400. Colorimetric results were measured using an Infinite M Plex (Tecan).
For the measurement of complex III and IV activity mitochondria were isolated from testis and brain stem using a mitochondria isolation kit for tissue (abcam). For WT a single testis (0.1 g ± 10%), for Parl−/− both testes (0.07 g ± 10%) and 0.07 g ± 10% of brain stem of each animal were homogenized in 2 mL of the provided isolation buffer. The homogenate was first centrifuged at 4 °C with 1000 × g for 10 min and then the supernatant was centrifuged at 4 °C with 12,000 × g for 15 min. The resulting pellet was washed twice by resuspending in isolation buffer and centrifugation. For the measurement of complex III the pelleted mitochondria were resuspended in 35–70 µL isolation buffer and protein concentration was measured using a Pierce BCA Protein Assay (Thermo Fisher Scientific). The activity of respiratory chain complex III was measured using a Mitochondrial Complex III Activity Assay Kit (Sigma-Aldrich) according to the instructions by adding 5 and 10 µg of protein in 1–2 µL of mitochondrial sample to the reaction mix on the 96-well plate. Finally, 6 µL of cytochrome c was added to each well and the change in absorption was measured every 30 s for 10 min at wavelength of 550 nm. Samples were issued in duplicates for each concentration. For each sample, a control with complex III inhibitor was performed by adding 2 µL of antimycin a to the mix. Furthermore, a negative control was performed by adding 2 µL of water instead of a sample to the reaction mix. The change in absorption was calculated within the linear range, divided by the protein concentration and the average of both concentrations was calculated. For the measurement of complex IV activity we used a Complex IV Rodent Enzyme Activity Microplate Assay Kit (abcam) by resuspending the pelleted mitochondria in 35 µL (testis) or 75 µL (brain stem) of solution 1 provided in the kit, measuring the protein concentration using a Pierce BCA Protein Assay (Thermo Fisher Scientific), diluting the concentration to 5.5 µg/µL with solution 1 and adding 1:10 detergent (final concentration 5 µg/µL. After 30 min of incubation on ice, followed by centrifugation, 5 µL (25 µg protein) of the supernatant were loaded into the wells of the provided assay microplate with 195 µL of solution 1. Samples and null controls (only solution 1) were tested in triplicates. After 3 h of incubation at RT and washing with solution 1, 200 µL of Assay solution containing reduced cytochrome c were added to the wells and the decrease in absorbance at 550 nm wavelength was measured every 2 min for 2 hours at 30°C. The decrease in absorbance was calculated within the linear range of the measurement (between 10 and 44 min). Mitochondria that were isolated in excess were used for Western blotting in the same way as described above for tissue lysats.
The ATP concentration of the testis and brain stem were measured using a luminescence ATP detection assay kit (Cayman). Three hundred fifty milligrams of tissue sample was homogenized in 400 µL of the provided sample buffer and deproteinized by adding 200 µL of 1 M perchloric acid to a 200-µL aliquot of the sample, followed by neutralization with 1 M potassium hydroxide. Deproteinized samples were used undiluted and at a dilution of 1:5 following the kit instructions. Luminescence was measured using an Infinite M Plex (Tecan).
Immunohistology and electron microscopy
Mice at the ages of 4 or 8 weeks were sacrificed by an overdose of carbon dioxide and post-mortem perfused through the left ventricle with phosphate-buffered saline (PBS), followed by perfusion with modified Bouin solution (4% paraformaldehyde [PFA], picric acid in PBS, pH 5, for immunohistology) or 6% glutaraldehyde in PBS (for electron microscopy). Testes were kept in the respective fixative overnight for post-fixation.
For immunohistological fluorescence staining, testes with epididymides were embedded in paraffin. Eight-micrometer-thick deparaffinized rehydrated sections were subjected to heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9), blocked for 1.5 h at RT using 10% bovine serum albumin (BSA, Bio West), and incubated with the indicated primary antibody at 4 °C overnight (see Table S1 for details). After washing with PBS, slides were incubated for 1.5 h at RT with the secondary antibody (DyLight 549, Goat anti-Rabbit, Vector) and counterstained with bisbenzimide (H33258, Sigma). Fluorescence images were acquired using an Eclipse 90i microscope (Nikon) equipped with an Intensilight C-HGFIE fiber illuminator (Nikon) and a D-Eclipse C1 laser scanning confocal microscope system (Nikon). Pictures of WT and Parl−/− were taken with the same parameters for each antibody.
To visualize apoptotic cells in paraffin-embedded testis sections, an ApopTag Peroxidase In Situ Apoptosis Detection Kit (Millipore), which applies the TUNEL method, was used according to the manufacturer’s instructions.
To test the functional integrity of the BTB, a biotin tracer (10 mg/mL EZ-Link Sulfo-NHS-LC-Biotin, Pierce) was injected into the interstitial space of the testes of freshly sacrificed mice, as described previously52. Injected testes were kept at RT for 30 min, and were then fixated in modified Bouin solution (pH 5) overnight and embedded in paraffin. To visualize the biotin tracer, 8-µm-thick deparaffinized rehydrated sections were blocked for 1 h with 5% BSA in PBS and incubated for 1 h at RT with DyLight 488 streptavidin (Vector).
For electron microscopy, testes were cut into slices measuring a few millimeters using a razor blade. The slices were washed in PBS, incubated for 1 h in 1% osmium tetraoxide (OsO4) in PBS, and dehydrated in an ethanol series. To prepare samples for scanning electron microscopy (SEM), slices were transferred to acetone using an increasing acetone series in ethanol, dried using a critical point dryer (CDP 030; Bal-Tec), fixed on a sample holder (sample holder for JEOL, Plano), and sputtered with platinum using a sputter-coater SCD 500 (Bal-Tec). Images were obtained using an SEM 7500 F (JEOL). For transmission electron microscopy (TEM), dehydrated samples were incubated in propylene oxide, followed by incubation and embedding in a SPURR medium. Semi-thin sections of 1-µm thickness were obtained and stained with toluidine blue. Ultra-thin sections were mounted on copper grids (200 G or 300 G mesh, Plano), contrasted, and analyzed using a JEM-1400 TEM (JEOL).
Testis cholesterol and serum steroid hormone levels
The cholesterol levels of 10 mg of dried testis samples were measured by flame-ionization detection following gas chromatographic separation (GC-FID) on a DB-XLB 30 m × 0.25 mm i.d. × 0.25 µm film (J&W Scientific Alltech) in a Hewlett-Packard 6890 Series GC-system (Agilent Technologies) equipped with an FID at the Institute of Clinical Chemistry and Clinical Pharmacology of the University Hospital Bonn as described in a previous study53.
For the measurement of steroid hormones, blood samples of 8-week-old mice were obtained postmortem (mice sacrificed for organ harvesting) through intracardial extraction from the right ventricle. Steroid hormones were determined at the Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University of Leipzig (Leipzig, Germany). 100 µL of serum was used for the simultaneous quantification of aldosterone, testosterone and progesterone using online solid phase extraction (SPE) LC-MS/MS as described in a previous study54. Lower limits of quantification ranged from 0.02 ng/mL (aldosterone), 0.03 ng/mL (testosterone) to 0.06 ng/mL (progesterone). General accuracy was 95–109% with between-run imprecision of ≤10%.
Leydig cell isolation and hCG stimulation
Leydig cells were isolated from 8-week-old WT and Parl−/− testes using a discontinuous Percoll (Cytiva) density gradient with slight alterations as described before55. Both testes of freshly sacrificed mice were washed, decapsulated and added to a collagenase IV solution with 1 mg/mL collagenase IV in medium for 15 min at 37 °C. To end digestion Medium containing fetal calf serum was added and the mix left to settle for a few minutes. The supernatant was filtered through a cell strainer (40 µm) and centrifuged for 5 min at 500 × g. The pellet was resuspended in 1 ml of HBSS and the solution layered on top of a discontinuous Percoll gradient with 60%, 50%, 40% and 30% Percoll in HBSS and centrifuged for 30 min at 800 × g at 4 °C. The Percoll fractions were isolated washed and centrifuged for 5 min at 500 × g. The cells were resuspended in 500 µL to 1 mL of DMEM + 10% FCS + 100 U/mL penicillin + 100 µg/mL streptomycin, counted using Typan blue (Gibco), and seeded with 1.5 × 105 cells per well on a 96-well plate. The cells were incubated at 5% CO2 and 37°C for 2 h, the medium was exchanged and the cells incubated. After 48 h the medium was taken off and the cells were stimulated with human chorionic gonadotropin (hCG) (Ferring) at a concentration of 10 mIU/mL for 2 h. To measure basal testosterone production one well per animal was not stimulated and medium without hCG was added. After 2 h the medium was removed, centrifuged and the supernatant snap frozen. Cells were washed with PBS, harvested using trypsin, centrifuged for 5 min at 500 × g and resuspended in PBS. A small droplet of the cell suspension was put on a microscope slide, left to dry and a 3-β-HSD-assay was performed as described by Liang et al.56 to assess the purity of Leydig cells. Dried cells were washed with PBS for 1 min, then the 3-β-HSD-assay-solution (0.5 mg Nitro blue Tetrazolium Chloride (NBT), 0.3 mg DHEA, 0.3 mL DMSO, 5 mg β-NAD, 4.75 mL PBS) was added and incubated for 30 min at 37 °C. The microscope slide was rinsed with PBS and left to dry. Purity was >85% with only minor infiltration of germ cells.
Testosterone measurement in cell culture medium
The testosterone level in the cell culture medium of the isolated Leydig cells was measured using a Testosterone ELISA Kit (Cayman, range: 3.9–500 pg/mL, sensitivity: ~6 pg/mL) following the manufacturer’s instructions. Samples were diluted at 1:50, 1:100 and 1:200 and tested together with standards in duplicates with the reaction mix on the pre-coated 96-well plate. After 2 h of incubation, wells were washed and Ellman´s reagent was added. Absorption was measured at 415 nm after 75 min incubation and testosterone levels were calculated using the standard curve.
Statistics and reproducibility
Statistical analyses were performed using GraphPad PRISM 9.0.0. Data with n ≤ 5 were statistically analyzed with a Mann–Whitney U Test or a Kruskal–Wallis Test with post hoc pairwise comparison (for p ≤ 0.05) and visualized in boxplots (center line: median, box limits: upper and lower quartiles, whiskers: 1.5 × interquartile range), with the significance level being indicated with asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001). Data with n > 5 were tested for normality using Kolmogorov–Smirnov tests and in case of normality (p > 0.05), data were subsequently analyzed using an unpaired t test or ANOVA with post hoc pairwise comparison (Tukey’s multiple comparisons test) for p ≤ 0.05. Data were visualized in bar charts, with error bars indicating standard deviation and asterisks indicating significance level (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).
For the measurement of testis weight, the average weight of both testes per animal was calculated, and for the analyses of tubule diameter, the average diameter of 10 seminiferous tubules of eight mice for each genotype was calculated.
To analyze the measurements of ATP and LH concentration, the measured concentration was considered in relation to the overall protein concentration of the homogenized samples (in the case of ATP concentration of the original sample before deproteinization) by dividing the measured ATP/LH level through the measured protein concentration (µg/mL).
The results of the RT-qPCRs were analyzed using the 2-ΔΔCT method57. ΔCT values were calculated by subtracting the CT value of the reference gene (Gapdh) from the CT value of the target gene. The ΔΔCT value was then calculated by subtracting the median ΔCT value of the WT from the ΔCT value of each WT and Parl−/− sample.
To analyze the protein expression of the isolated mitochondria using Western blot, the density of bands was measured using ImageJ. Relative protein expression was calculated by dividing the values of the target protein by the value of a reference protein (HSP60, aconitase 2 (ACO2)). To calculate the expression relative to the WT control, all values were divided by the median of the WT control group.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.