Fig 2: (A) Immunocytochemistry of KISS1 and KISS1R in Bouin’s fixed C57BL/6 testis sections (7 µm). The KISS1 and KISS1R protein localization in Leydig cells was indicated by black and white arrowheads, respectively. Scale bar: 50 µm. (B–G) Differential expression analysis of KissR and cytoskeletal–nucleoskeletal pathway mediator mRNAs in mice testes in vitro treated with different doses of Kp-10 (0.01 µM, 0.1 µM, and 1 µM), by qRT-PCR. (B) Kiss1R, (C) Ankrd31, (D) Spectrin, (E) ß-Actin, (F) Nesprin2, and (G) Sun2 expression levels were normalized using Rp18S as a housekeeping gene and expressed as normalized fold expression (n.f.e.), relatively to the CTRL group. All data are reported as mean value ± S.E.M; *p < 0.05; **p < 0.01. Western blot analysis of (H) KISS1R, (I) ANKRD31, (J) SPECTRIN-a-II, (K) ß-actin, (L) F-actin, (M) NESPRIN2, and (N) SUN2 proteins levels in mice testes in vitro treated with different doses of Kp-10 (0.01 µM, 0.1 µM, and 1 µM). Signals were quantified by the densitometry analysis and normalized to Ponceau Red (Pon.S). Data are expressed in O.D. values as fold change (O.D. fc), relatively to the CTRL group, and reported as mean ± SEM; *p < 0.05; **p < 0.01.

Fig 3: (A,B) IP in murine primary Leydig cells. Total proteins collected from murine primary Leydig cell cultures were immunoprecipitated using (A) KISS1R and (B) ANKRD31 antibodies, respectively. Protein interaction among KISS1R, ANKRD31, and F-actin was detected by Western blot analysis. (C) IP in murine primary Leydig cells in vitro treated with Kp-10 alone (0.1 µM) or in combination with the specific antagonist Kp-234 (1 µM) using KISS1R antibody. Protein interaction among KISS1R, ANKRD31, and F-actin was detected by Western blot analysis. (D) Immunofluorescence analysis of F-actin, using phalloidin staining (green) in murine primary Leydig cells in vitro treated with Kp-10 alone (0.1 µM) or in combination with the specific antagonist Kp-234 (1 µM). Nuclei were labeled with TO-PRO3 iodide (blue). Scale bar: 37.5 µm. (E) Quantitative immunofluorescence analysis: F-actin signals were normalized against nuclei number, expressed in SUM(I) values and reported as mean ± SEM; experimental groups with statistically significant differences (p < 0.01) were indicated with different letters; the experimental groups without statistically significant differences were indicated with the same letter. (F) Analysis of TT content (as ng/ml) in culture media of Leydig cells in vitro treated with Kp-10 alone (0.1 µM) or in combination with the specific antagonist Kp-234 (1 µM). All the data were reported as mean ± SEM; **p < 0.01. Experimental groups with statistically significant differences (p < 0.01) were indicated with different letters. (G) Western blot analysis of CYP19 in murine primary Leydig cells in vitro treated with Kp-10 alone (0.1 µM) or in combination with the specific antagonist Kp-234 (1 µM). Signals were quantified by the densitometry analysis and normalized to Ponceau Red (Pon.S). Data are expressed in O.D. values as fold change (O.D. fc), relatively to the CTRL group, and reported as mean ± SEM; experimental groups with statistically significant differences (p < 0.01) were indicated with different letters.