C57BL/6 male mice (Charles River Laboratory, Lecco, Italy) were used in this study. All animals were housed as three per cage under controlled illumination (12 h light/dark cycle; light on 6:00 a.m.) and standard environmental conditions (ambient temperature 20–22°C, humidity 55%–60%) and were maintained on a standard pellet diet with free access to water, before the beginning of experimental procedures. The number of the enrolled animals was determined by the parameters adopted for the G*Power analysis required to get the permission for in vivo experiments, which is suggested by the legal entity giving the permission. For experimental procedures, adult males (3–5 months) were sacrificed under anesthesia, by cardiac perfusion with PBS (pH 7.6), to clean peripheral tissues from blood contaminants, or by cervical dislocation, depending on the experimental designs. Animals were placed in a plexiglas chamber with 4% isoflurane (Iso-Vet, Piramal Healthcare, United Kingdom) for 5 min and were sacrificed when fully sedated, as measured by a lack of heartbeat and active paw reflex. Testes were rapidly removed and collected depending on the experimental procedure, as described later.

In addition, wild-type (WT) male mice and males carrying Ankrd31 null mutation (Ankrd31 ^−/−) (Manfrevola et al., 2021b) were used in this study. Heterozygous mice were bred on a C57BL/6 background before generating WT and Ankrd31 ^−/− male mice. For experimental procedures, WT and Ankrd31 ^−/− adult males (3–5 months) were sacrificed, under anesthesia, by cervical dislocation. Testes and blood samples were rapidly removed and collected depending on the experimental procedures.

Kp-10 (Metastin 45–54, H-YNWNSFGLRF-NH2) of human origin and Kp-234, a specific KISS1R antagonist (Roseweir et al., 2009), were purchased from DBA Italia (Milan, Italy). Kp-10 of human origin was chosen on the basis of its high homology with the murine peptide sequence. Cytochalasin D (C8273) was obtained from Sigma-Aldrich (Milan, Italy). The drugs were dissolved in dimethylsulfoxide (DMSO), according to the manufacturer’s instructions. NB4 collagenase was obtained from Serva (Heidbergh, Germany).

Testes collected from WT and Ankrd31 ^−/− mice (n = 5 for each group) were fixed overnight in Bouin’s solution, dehydrated in ethanol, cleared in xylene, and embedded in paraffin using standard procedures. Microtome serial sections (7 μm thick) were cut and processed for hematoxylin/eosin (H&E) staining and immunocytochemistry staining analyses. For H&E staining, the sections were deparaffinized and processed using standard procedures. For immunocytochemistry staining, testes sections were deparaffinized, rehydrated, and permeabilized with PBS pH 7.4 containing 0.1% Triton X-100. A citrate buffer of 0.01 M (pH 6.0) was used for antigen retrieval. After blocking with PBS containing 5% BSA and normal goat serum (diluted 1:5), sections were incubated with anti-KISS1 antibody (PA5-50513, Invitrogen; diluted 1:100), anti-KISS1R antibody (PA5-96221, Invitrogen; diluted 1:100), anti-F-actin antibody (MA1-80729, Invitrogen; diluted 1:100), anti-H3K14ac antibody (703894, Invitrogen; diluted 1:100), and anti-StAR antibody (sc-166821, Santa Cruz, diluted 1:100) overnight at 4°C. Immunoreactivity was revealed using the avidin/biotin complex system and H₂O₂/DAB as the substrate/chromogen. The specificity of immunoreactions is routinely checked by omitting primary antibodies. The histological observations and analysis were carried out under a light microscope (Leica CTR500, Leica Microsystems Inc., Milan, Italy), and images were captured using a high-resolution digital camera (Leica DC300F).

WT (n = 5) and Ankrd31 ^−/− (n = 5) male mice (3 months old) were injected with hCG (15 IU/Kg; Chorulon Grovet) intraperitoneally. After 2 h from hCG injection, blood samples were collected and plasma was obtained by centrifugation at 1,000× g for 10 min. Plasma samples were stored at −20°C for further testosterone dosage.

Plasma TT levels of WT (n = 5) and Ankrd31 ^−/− (n = 5) male mice, at basal conditions and following hCG injection, were measured by a competitive enzyme immunoassay (EIA) designed by the Phenotyping Endocrinology Laboratory (Research unit: Physiology of Reproduction and Behavours, Nouzilly, France). The sample size used for the assay was 8 µL of undiluted sample. The minimal detectable TT concentration was 0.15 ng/ml, and all samples were analyzed in the same assay. The intra- and inter-assay coefficients of variation were 4.7% and 7.1%, respectively. All determinations were made in triplicate for each plasma sample analyzed.

C57BL/6 testes (n = 5 for each experimental group) were incubated in PBS for 90 min at room temperature (RT), with vehicle (0.005% DMSO; control group, CTRL) or with Kp-10 at 0.01 μM, 0.1 μM, and 1 μM. After treatment, the testes were placed at −80°C for following molecular investigations.

Leydig cells were isolated as previously described by Sun et al. (2011). In brief, testes from adult mice (n = 5) were harvested, and tunica albuginea was removed. Then, testes were digested at 37°C for 15 min in 0.03% collagenase NB4 in a thermomixer imposing 150 rpm vibration. After this first digestion, the supernatant was discarded. Then, testes were incubated again in 0.03% collagenase NB4 in the thermomixer for 15 min, lowering the vibration speed to 130 rpm. Isolated cells were counted and approximately 1 × 10⁵ cells per testis were obtained. Isolated cells were cultured in low glucose DMEM with 10% of fetal bovine serum (FBS) plating them in culture plates (60 mm or 35 mm culture dish Falcon) for molecular analysis and in eight well chamber slides (Ibidi) for immunofluorescence experiments. STAR staining was carried out to identify the cells and to assess their purity (data not shown). When the Leydig cells were grown to 90% confluence were treated, when indicated, with Kp-10 (0.1 µM), Kp-234 (1 µM), and cytochalasin D (10 µM) alone or in combination with Kp-10. Cytochalasin D and Kp-234 were added 30 min before Kp-10 treatment.

For in situ analyses, Leydig cells, cultured in eight well chamber slides, were fixed in 4% paraformaldehyde in PBS (pH 7.4) at 4°C overnight. To detect F-actin and H3K14ac, immunofluorescence experiments were performed. Fixed cells were permeabilized in PBS supplemented with 1% BSA and 0.1% Triton for 2 h and then quenched with 5% donkey serum in PBS/BSA/Triton. Samples were then incubated overnight with anti-H3K14ac antibody diluted 1:100 (703894, Invitrogen Life Technologies, Paisley, United Kingdom). Then, samples were washed three times in PBS/BSA/Triton for 30 min and incubated with the appropriate secondary antibody: Cy5 conjugated anti-rabbit IgG was used to detect H3K14ac immunocomplexes (Jackson Immuno Research, Cambridge, United Kingdom). TO-PRO3 iodide fluorescent dye 642/661 (1:5000 in PBS, Invitrogen Life Technologies, Paisley, United Kingdom) was used for nuclei staining. As a negative control, the primary antibody was omitted. For the detection of F-actin, a FITC-conjugated phalloidin (Invitrogen, Life Technologies, Paisley, United Kingdom) was used.

Immunofluorescence experiments were analyzed using a Leica confocal microscope (Laser Scanning TCS SP2 equipped with Kr/Ar and He/Ne lasers, Mannheim, Germany). Laser lines were 488, 543, and 633 nm for FITC, Cy5, and TO-PRO3 excitation, respectively. The images were scanned under a ×20 or ×40 oil immersion objectives. To perform quantitative analysis of fluorescence, optical spatial series with a step size of 1 µm were recovered with fixed laser intensities, and the sum of fluorescence intensity (SUM(I)) was determined in maximum projection image of each series using Leica confocal software. Three independent experiments in triplicate were analyzed.

Culture media of Leydig cells in vitro treated with Kp-10 (0.1 µM) alone or in combination with Kp-234 (1 µM) were collected to perform TT determination. TT was extracted from cell culture media using methanol as an organic solvent (liquid/liquid extraction) and purified by solid phase extraction (SPE) using specific cartridges for steroids (AFFINIMIP, Affinisep, France). The procedure described by Errico et al. (2019) was modified specifically for cell culture media including the quality control system used to monitor method performance and to prevent assay contamination. The LC-MS/MS analysis was performed using a Dionex UltiMate3000 HPLC system (Thermo Fisher Scientific Inc, Monza, Italy), coupled to an ESI-triple quadrupole mass spectrometer (API 2000, Sciex, Germany). A Kinetex F5 (×100 4.6 mm, 2.6 µm) column (Phenomenex, Italy) was used for reversed-phase separations. Chromatographic separation and instrumental parameters were reported in Errico et al., 2019. The analyte identification was based on multiple reactions monitoring in a positive mode. The TT was specifically identified not only by the retention time but also by monitoring the following ion transitions: m/z 291.3 → m/z 97.2 (quantifier) and m/z 291.3 → m/z 108.9 (qualifier). The linearity of the detector response was verified over the concentration range 0.100–50 ng/ml. All samples were analyzed in triplicate, for each experimental group, with relative standard deviations (RSDs) less than 13%. HPLC grade reagents, including ultrapure water, acetonitrile (ACN), and methanol (MeOH), were purchased from Romil (ROMIL Ltd., United Kingdom). TT was purchased from Merch (Germany). AFFINIMIP cartridges were purchased from Affinisep (France).

The TRIzol^® reagent (Invitrogen Life Technologies, Paisley, United Kingdom) was used to extract total RNA from C57BL/6 testes in vitro treated with Kp-10 (0.01 μM, 0.1 μM, and 10 μM) and from primary murine Leydig cells in vitro treated with Kp-10 (0.1 μM) and cytochalasin D (10 μM) alone or in combination with Kp-10 (0.1 μM). In brief, the testes were homogenized in the TRIzol reagent and incubated for 5 min at 20°C. Then, 0.2 ml chloroform/ml Trizol reagent were added, and the samples were centrifuged at 12,000× g for 15 min at 4°C. The aqueous phase was transferred to a fresh tube and total RNA was precipitated by mixing with isopropyl alcohol (0.5 ml/ml Trizol reagent) and 1 μl of glycogen (20 mg/ml). After centrifugation at 12,000× g for 10 min at 4°C, the RNA pellet was washed with 75% ethanol, centrifuged at 7,500× g for 10 min at 4°C, and dissolved in DEPC-H₂O. Total RNAs were assessed with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States) to quantify concentration (ng/μl) and purity (260/280 and 260/230 ratios). Then, RNA aliquots (10 μg) were treated with 2U DNase I (RNase-free DNase I, Ambion, Thermo Fisher Scientific, Massachusetts, United States) to remove potential contamination of genomic DNA and finally preserved at −80°C until the next step.

According to the manufacturer’s instructions, a kit containing quantitative real-time polymerase chain reaction (qRT-PCR) enzyme mix and an Evagreen qPCR Mastermix (Applied Biological Materials Inc., Richmond, Canada) was used for gene expression analysis in i) testes in vitro treated with Kp-10 (0.01 μM, 0.1 μM, and 10 μM) (n = 5 animals for each experimental group) and in ii) primary murine Leydig cells in vitro treated with Kp-10 (0.1 μM) and cytochalasin D (10 μM) alone or in combination with Kp-10 (0.1 μM) (n = 5 samples for each experimental group). A concentration of 50 ng of total RNA was used for all reactions on a CFX-96 Real-Time Polymerase Chain Reaction (PCR) System (Biorad, Milan, Italy). A negative control, without RNA, was included. The qRT-PCR in triplicate from each experimental group was analyzed. A gene expression analysis, corrected for PCR efficiency, and normalized toward the reference gene (Rp18S), was performed by CFX Manager software (Biorad, Milan, Italy). Normalized fold expression (n.f.e) of mRNAs was calculated by applying the 2^–ΔΔCT method.

Primers to amplify selected RNAs were designed through the online tool Primer-BLAST1. Primers for mouse genes are shown in Table 1.

Total protein extraction was performed for i) testes in vitro treated with Kp-10 (0.01 μM, 0.1 μM, and 10 μM) (n = 5 animals for each experimental group), ii) primary murine Leydig cells in vitro treated with Kp-10 (0.1 μM) alone or in combination with the specific antagonist Kp-234 (1 μM) (n = 5 samples for each experimental group), and iii) primary murine Leydig cells in vitro treated with Kp-10 (0.1 μM) and cytochalasin D (10 μM) alone or in combination with Kp-10 (0.1 μM) (n = 5 samples for each experimental group). The samples were separately homogenized in RIPA buffer [PBS, pH 7.4, 10 mM of dithiothreitol, 0.02% sodium azide, 0.1% SDS, 1% NP-40, and 0.5% sodium deoxycholate, in the presence of protease inhibitors (10 μg/ml of leupeptin, aprotinin, pepstatin A, chymostatin, and 5 μg/ml of TPCK)] and sonicated three times for 30 s bursts, each at 60 mW. Proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (GE Healthcare, Milan, Italy) at 280 mA for 2.5 h, at 4°C. The filters were treated for 2.5 h with blocking solution [5% non-fat milk, 0.25% Tween 20 in Tris-buffered saline (TBS, pH 7.6)] and incubated with different primary antibodies in TBS-milk buffer (TBS pH 7.6, 3% non-fat milk) overnight, at 4°C. The filters were washed in 0.25% Tween 20–TBS and incubated with secondary antibodies diluted 1:1,000 in TBS-milk buffer and then washed again. An enhanced chemiluminescence–Western blotting detection system (Amersham ECL Western Blotting Detection Reagent, cod: RPN2106, GE Healthcare, Milan, Italy) was used to detect the immune complexes.

Antibodies and relative dilutions are reported in Table 2. The specificity of the immunoreactions was routinely checked by omitting all primary antibodies used in this study (data not shown). Western blot experimental triplicates from each experimental group were quantified by densitometry analysis, adjusted relatively to Ponceau S staining, and reported as O.D. fold change (mean ± SEM).

For IP, primary murine Leydig cells in vitro treated with Kp-10 (0.1 μM) alone or in combination with the specific antagonist Kp-234 (1 μM) were lyzed in RIPA buffer, in the presence of protease inhibitors (10 μg/ml of leupeptin, aprotinin, pepstatin A, chymostatin, and 5 μg/ml of TPCK), sonicated three times for 30 s bursts, each at 60 mW, and then incubated on ice for 30 min. After centrifugation at 20,000× g for 30 min at 4°C, the protein supernatant was collected. A concentration of 500 μg of supernatant proteins from each sample was incubated with 2 μg of KISS1R antibody (IP-KISS1R), ANKRD31 antibody (IP-ANKRD31), or IgG as negative control (12370, Sigma-Aldrich, Milan, Italy), under rotary agitation, overnight at 4°C. Afterward, Protein A/G PLUS Agarose Beads (sc-2003, Santa Cruz Biotechnology, Cambridge, United Kingdom) were added to each sample and incubated overnight under rotary agitation at 4°C. After bead incubation, samples were washed three times (3,000× g for 3 min at 4°C) in TBS pH 7.6 and boiled in Laemmli sample buffer for 10 min to be later analyzed by SDS-PAGE.

ANOVA followed by Student’s t-test (for two independent group comparisons) and Tukey test (for multi group comparison) was used to identify groups having different mean. Differences with p < 0.05 were considered statistically significant. Data were expressed as the mean ± SEM from at least five independent animals for each genotype or experimental group. For qRT-PCR, Western blot, LC-MS/MS, and EIA assay, triplicates from five animals/genotypes or experimental groups each were considered.

In order to study the effects of Kps in the regulation of the Kp/KISS1R system and the cytoskeletal–nucleoskeletal pathway in Leydig cells, we evaluated i) the expression of the Kp/KISS1R system in WT mouse testis, by an immunocytochemistry analysis, and ii) the expression of several cytoskeletal–nucleoskeletal mediators, in testes in vitro treated with different doses of Kp-10 (0.01 µM, 0.1 µM, and 1 µM), by qRT-PCR. Results showed a striking KISS1 and KISS1R protein localization in Leydig cells (Figure 1A). Interestingly, higher expression levels of Kiss1R, at all the chosen doses of Kp-10, were observed in comparison to the CTRL group (Figure 1B) (p < 0.01). In addition, a significant increase in Ankrd31, Spectrin, and β-Actin expression levels was also observed at all the chosen doses of Kp-10, in comparison to the CTRL group (Figures 1C–E) (p < 0.05; p < 0.01), whereas lower expression levels of Nesprin2 and Sun2 were observed following Kp-10 in vitro treatment (Figures 1F,G) (p < 0.01).

Western blot analysis was carried out to investigate the testicular protein levels of KISS1R receptor and cytoskeletal–nucleoskeletal modulators, following Kp-10 in vitro treatment. As reported, data showed that all the analyzed proteins were significantly increased following Kp-10 treatment. In details, a significant increase of testicular KISS1R, ANKRD31, and SPECTRIN protein amount was observed at all the chosen doses of Kp-10, relatively to the CTRL group (Figures 1H–J) (p < 0.01). In agreement, the levels of cytoskeletal modulators, as β-actin and F-actin, were higher in testes in vitro treated with Kp-10 than the CTRL group (Figures 1K,L) (p < 0.05; p < 0.01) and, finally, a significant increase of NESPRIN2 and SUN2 protein levels was also observed at all the doses, relatively to the CTRL group (Figures 1M–N) (p < 0.05; p < 0.01), suggesting that Kp-10 in vitro treatment positively modulates the expression of both KISS1R receptor and cytoskeletal–nucleoskeletal pathway modulators, having Leydig cells as the main target.

Considering that in mouse testis KISS1R is expressed in Leydig cells (Anjum et al., 2012; Salehi et al., 2015), as confirmed by our immunocytochemistry analysis, we evaluated a possible effect of Kps on the expression of key modulators of steroidogenesis. Results showed a significant increase of Lhr and Hsd3b expression levels at the Kp-10 doses of 0.1 and 1 µM (p < 0.01), (Figures 2A,B), in comparison with the CTRL group, whereas no effect was observed at the Kp-10 dose of 0.01 µM (Figures 2A,B). Similar results were observed for Star and Sf1 (Figures 2C,D).

Western blot analysis was carried out to investigate a possible responsiveness of epigenetic markers following Kp in vitro treatment. As showed, relatively to the CTRL group, a significant increase of H3K14ac protein levels, chosen as a marker of active gene transcription, was observed at Kp-10 doses of 0.1 µM and 1 µM (p < 0.01), whereas no effect was observed at the dose of 0.01 µM (Figure 2E). Analogously, testicular in vitro treatment with the doses 0.1 and 1 µM of Kp-10 significantly decreased protein levels of HDAC1, HADAC2, and HDAC4 (p < 0.01) than the CTRL group (Figures 2F,G), whereas no effect was observed at the dose of 0.01 µM for all HDAC types investigated, suggesting that Kps might positively regulate Leydig cell gene expression modulating epigenetic markers.

In order to demonstrate that the Kp/KISS1R system could regulate Leydig cell functions and gene expression via ANKRD31, and in turn by the cytoskeletal–nucleoskeletal pathway, we carried out immunocytochemistry and molecular analyses in Ankrd31 ^−/− mouse testes, chosen as putative model of cytoskeletal disruption. A histological analysis performed by H&E staining on testicular sections of both WT and Ankrd31 ^−/− showed an impairment of interstitial environment in Ankrd31 ^−/− compared to WT (Figure 3A), suggesting that the loss of ANKRD31 protein could affect Leydig cell functions. Interestingly, the immunocytochemistry analysis showed a lower signal of both KISS1 and KISS1R in Ankrd31 ^−/− Leydig cells than WT, confirming a deregulation of the Kp/KISS1R system (Figures 3B,C). In addition, the immunocytochemistry analysis of F-actin, H3K14ac as well as STAR evidenced a strong signal reduction of all markers analyzed in Ankrd31 ^−/− compared to WT Leydig cells (Figures 3D–F). To confirm Kp/KISS1R system deregulation, the Western blot analysis of KISS1R was carried out in WT and Ankrd31 ^−/− testis. As reported, a significant reduction of KISS1R protein content was observed in Ankrd31 ^−/− in comparison to WT testis (p < 0.01) (Figure 3G). In order to assess if the deregulation of the Kp/KISS1R system observed in Ankrd31 ^−/− Leydig cells occurred due to their compromised functions, we evaluated the expression of key modulators of steroidogenesis by the Western blot analysis. The results showed a significant decrease of LHR, HSD3β, STAR, and SF1 protein contents in Ankrd31 ^−/− in comparison to WT (p < 0.01), (Figures 3H–K). In agreement, plasma TT levels were significantly lower in Ankrd31 ^−/− than WT (p < 0.01) in both basal conditions as well as following hCG stimulation (Figure 3L), confirming that i) Ankrd31 ^−/− Leydig cells possessed an impaired ability to respond to LH stimulation and ii) the deregulation of Kp/KISS1R system and of steroidogenesis modulators negatively affected TT production in Ankrd31 ^−/− mice.

In order to investigate if a stimulation of KISS1R may be able to regulate Leydig cell gene expression, via the cytoskeletal–nucleoskeletal pathway, IP experiments were carried out in murine primary Leydig cell cultures to show a physical interaction among KISS1R, ANKRD31, and F-actin.

We first immunoprecipitated KISS1R from total proteins extracted from Leydig cells (IP-KISS1R), followed by immunoblotting with KISS1R, ANKRD31, and F-actin antibodies (Figure 4A). The results showed stronger KISS1R, ANKRD31, and F-actin signals in IP-KISS1R as compared with a significantly weaker control signals, suggesting a protein complex formation among KISS1R, ANKRD31, and F-actin in Leydig cells (Figure 4A). Accordingly, the immunoprecipitation of ANKRD31 (IP-ANKRD31) from total proteins extracted from Leydig cells and the immunoblotting with KISS1R, ANKRD31, and F-actin antibodies confirmed the existence of the protein complex (Figure 4B).

In addition, murine primary Leydig cell cultures were in vitro treated with Kp-10 alone (0.1 µM) or in combination with the specific antagonist Kp-234 (1 µM), with the aim to investigate the cytoskeletal–nucleoskeletal responsiveness to the Kp/KISS1R system activation in Leydig cells. Following the treatment, total Leydig cell protein extracts, derived from the three experimental groups (CTRL, Kp-10, and Kp-10+Kp-234), were used for KISS1R protein IP experiments. Immunoblotting with KISS1R, ANKRD31, and F-actin antibodies showed KISS1R, ANKRD31, and F-actin signals in IP-KISS1R carried out in all experimental groups (Figure 4C). Interestingly, results evidenced a strong increase of protein interaction among KISS1R, ANKRD31, and F-actin following Kp-10 treatment (Figure 4C). This increase was significantly counteracted by Kp-234 treatment and was dependent on the variations of total protein content, as confirmed by the analysis of input samples (total lysates isolated before the IP) (Figure 4C). Data suggested that Kp effectively increases the expression of KISS1R, ANKRD31, and F-actin and, consequentially, the formation of protein complex in Leydig cells.

Phalloidin staining was carried out in Leydig cells in vitro treated with Kp-10 alone or in combination with the specific antagonist Kp-234, in order to investigate the effect of Kp/KISS1R system activation on actin polymerization (Figure 4D). As showed, relatively to the CTRL group, a strong increase in phalloidin staining was observed in Leydig cells treated with Kp-10, whereas the signal intensity in the combined treatment Kp-10 + Kp-234 was similar to the control (Figure 4D). Consistently, phalloidin immunofluorescence quantification, relative to the CTRL group, confirmed a significant increase of the signal in Leydig cells treated with Kp-10 (p < 0.01); this effect was efficiently counteracted by Kp-234 (Figure 4E), suggesting that the Kp/KISS1R system activation in Leydig cells promoted an active F-actin synthesis, in addition to the enhancing of the protein complex among KISS1R, ANKRD31, and F-actin.

In order to show if KISS1R, ANKRD31, and F-actin protein complex may affect Leydig cell biological functions, TT levels were quantified in culture media of Leydig cells in vitro treated with Kp-10 alone (0.1 µM) or in combination with Kp-234 (1 µM) by the LC-MS/MS analysis. Interestingly, TT levels significantly decreased following Kp-10 treatment (p < 0.01), relatively to the CTRL group, and such an effect was significantly counteracted by Kp-234 treatment (Figure 4F). In addition, a significant increase of CYP19 protein content was observed after Kp-10 treatment (p < 0.01), in comparison to the CTRL group, by Western blot (Figure 4G). This effect was counteracted by Kp-234 (Figure 4G), suggesting that Kp/KISS1R system activation in Leydig cells may enhance testosterone conversion to estrogens.

With the purpose to demonstrate that Kps could regulate gene expression in Leydig cells, via the activation of the cytoskeletal–nucleoskeletal pathway, murine primary Leydig cell cultures were in vitro treated with Kp-10 (0.1 µM), cytochalasin D (10 µM), and, finally, with Kp-10 in combination with cytochalasin D. Following pharmacological treatments, co-immunofluorescence analysis was carried out to investigate the responsiveness of F-actin and H3K14ac to the different experimental conditions (Figure 5A).

Accordingly, Kp-10 induced a strong increase of F-actin signal, as previously reported, whereas a complete disaggregation of F-actin was observed following cytochalasin D treatment. In addition, Kp-10 was not able to effectively counteract the depolymerization effect of cytochalasin D on F-actin filaments (Figure 5A). Interestingly, H3K14ac immunofluorescence signal was increased in Leydig cells treated with Kp-10, whereas a weak signal was detected in Leydig cells treated with cytochalasin D alone or in combination with Kp-10 (Figure 5A).

Immunofluorescence quantification data, relatively to the CTRL group, confirmed the significant increase of F-actin staining in Leydig cells treated with Kp-10 (p < 0.01) and its reduction after cytochalasin D alone or in combination with Kp-10 (p < 0.01) (Figure 5B). In addition, H3K14ac immunofluorescence quantification showed the same trend observed for F-actin. In details, a significant increase of H3K14ac amount was observed in Leydig cells treated with Kp-10 (p < 0.01) and, consistently, a strong reduction occurred when Leydig cells were treated with cytochalasin D alone or in combination with Kp-10 (p < 0.01) (Figure 5C), suggesting that Kp/KISS1R system activation modulates the epigenetic landscape of Leydig cells via F-actin synthesis.

To strongly demonstrate this hypothesis, Western blot analysis of F-actin, cytoskeletal–nucleoskeletal mediators, as NESPRIN2 and SUN2, and H3K14ac was carried out in murine primary Leydig cells in vitro treated with Kp-10 and cytochalasin D alone or in combination with Kp. For F-actin, results showed a significant increase following Kp-10 treatment (p < 0.01) and a significant reduction following cytochalasin D treatment alone or in combination with Kp-10 (p < 0.01), in comparison with the CTRL group (Figure 5D). In addition, both NESPRIN2 and SUN2 showed higher protein levels following Kp-10 treatment and lower protein levels following cytochalasin D treatment alone or in combination with Kp-10, than the CTRL group, respectively (p < 0.01) (Figures 5E,F). Finally, relatively to the CTRL group, a significant increase of H3K14ac protein levels was observed in Leydig cells treated with Kp-10 (p < 0.01), and a significant reduction was observed when Leydig cells were treated with cytochalasin D alone or in combination with Kp-10 (p < 0.01) (Figure 5G).

With the aim to highlight that Kps, via the cytoskeletal–nucleoskeletal pathway activation, could enhance H3K14ac and, in turn, Leydig gene expression, the expression analysis of several markers was carried out by qRT-PCR. The results showed a significant increase of Lhr, Hsd3b, Star, and Sf1 expression levels following Kp-10 treatment (p < 0.01), in comparison with the CTRL group (Figures 5H–K), whereas a significant reduction was observed following cytochalasin D treatment, alone or in combination with Kp-10 (p < 0.01) (Figures 5H–K), suggesting that Kps enhance Leydig cell gene expression increasing key modulators of the cytoskeletal–nucleoskeletal pathway and, then, epigenetic markers.