The Remodeling of Mitochondrial-Endoplasmic Reticulum Contacts by Omega-3 Fatty Acids Mitigates Dietary Advanced Glycation End Product-Driven Sertoli Cell Senescence and Oligoasthenozoospermia

Abstract

Dietary components or patterns have been shown to affect male fertility. The increasing intake of processed foods rich in advanced glycation end products (AGEs) may threaten spermatogenesis. However, the key cell type affected by AGEs in spermatogenic microenvironment remains unspecified. Furthermore, given that subcellular organelle interactions, particularly communications between mitochondria and endoplasmic reticulum (ER), are of paramount importance in male fertility, it is worthwhile to investigate dynamic changes of mitochondria-ER contacts (MERCs) in AGE-driven spermatogenesis dysfunction. In this study, we found that serum AGEs levels increased in patients with oligoasthenozoospermia (OAZ), which was accompanied by decreased inhibin B levels, leading us to explore the effect of AGEs on Sertoli cells. In vivo experiments revealed that AGEs-rich diet disrupted spermatogenesis and induced Sertoli cell senescence and dysfunction in mice. We further confirmed that AGEs elicited an increase in MERCs, as well as ER stress and mitochondrial dysfunction in Sertoli cells. Omega-3 polyunsaturated fatty acids (omega-3), which are a category of dietary supplements with the potential to improve male fertility, were employed in the rescue experiment. We demonstrated that omega-3 mitigate dietary AGE-induced Sertoli cell senescence and OAZ via the remodeling of MERCs, highlighting the AGE-RAGE axis as a potential target for treating male infertility.

Keywords: Advanced glycation end products; Male infertility; Mitochondria‒endoplasmic reticulum contacts; Omega-3 fatty acids; Receptor for AGE; Sertoli cell.

Figure 1

Serum AGEs levels are significantly higher in patients with OAZ. (A) Serum AGEs levels in CTL and OAZ patients were analyzed by ELISA. (B-C) Univariate Logistic regression analysis (blue lines) was performed to determine whether serum AGEs is a risk factor for OAZ occurrence. Multivariate Logistic regression analysis (red lines) assessed whether serum AGEs, FSH, LH, T, INHB, and age are OAZ risk factors. A ROC curve was plotted with an AUC calculated. The forest plot from the Multivariate Logistic regression analysis showed the Odds Ratios (OR) and 95% Confidence Intervals (CI) for each factor. An unpaired t-test was used for two - group comparisons. A two - sided p - value < 0.05 was statistically significant, with p < 0.01 defined as highly significant (**).

Figure 2

Omega-3 mitigates AGEs effects on spermatogenesis and testicular senescence. (A) Animal Experimental Design: All animals were randomly divided into three groups: a control group (CTL), an advanced glycation end product-rich diet group (dAGEs) for 16 weeks, and a group fed an AGEs-rich diet supplemented with omega-3 PUFAs for the last 4 weeks (dAGEs + Omega-3). (B, C) Concentrations of AGE in sera(B) and testis(C) were analyzed by ELISA. (D-F) The mRNA levels (D) and the protein expression(E) of RAGE in mice testis. Protein expression was quantified by ImageJ and normalized (F). (G, H) Sperm concentration (G) and sperm progressive motility (PR) (H) of the mice after 16 weeks of treatment. (I) Representative photographs of testicular (upper and middle) and epididymal tail (lower) morphology with HE staining. Scale bars: for testis pictures, 100 μm for the original picture and 25 μm for the enlarged picture; for epididymal tail picture, 50 μm. Spermatogonia (SPG), Spermatocytes (SPC), Round Spermatids (RS), Elongated Spermatids (ES) and Sertoli Cells (SC) are indicated. (J-K) Diameter assessment and epithelial thickness quantification of each seminiferous tubule across different groups. (L-N) The expression of P16 (upper) and P21 (lower) proteins in mouse spermatogenic tubules was assessed by immunohistochemistry. Scale bars: 50 μm (M, N) Quantitative analysis using Average Optical Density (AOD) to assess the expression levels of proteins in tissue sections and normalized. (O) The levels of SASP factors in mouse testes were displayed using a heatmap. (P, Q) Representative images of ROS staining (P) and TUNEL staining (Q) in mice testicular spermatogenic tubules. Scale bars: 50 μm. Statistical analysis between multiple groups was performed by one-way ANOVA. A two-sided p-value < 0.05 was considered to be statistically significant. The level of significance defined as p < 0.05 (*), p < 0.01 (**), p < 0.005(***), p < 0.001 (****).

Figure 3

Omega-3 alleviates AGE-induced Sertoli cell injury and dysfunction in vivo. (A) Serum inhibin B levels were measured in CTL and OAZ patients. (B) The mRNA levels of Sertoli cell markers of mouse testis. (C) Immunohistochemistry staining of SOX9 protein expression. Scale bars: 50 μm. (D) Statistical analysis of the number of SOX+ cells in each seminiferous tubule across different groups. (E-F) Representative fluorescence images (E) of BTB integrity detected by the biotin tracer assay. The white asterisks (*) indicate the permeation of biotin into the seminiferous lumen. Scale bars: 100 μm. Bar graph (F) illustrating the extent of BTB damage, which was calculated randomly from 50-60 seminiferous tubules for each group. (G) The ultrastructure of tight junctions was observed using transmission electron microscopy. White arrows indicate the tight junctions of Sertoli cells and white asterisks denote discontinuous and loose structures. (H, I) The protein expression of ZO-1 and Occludin in mice testis was detected using Western blotting (H) and quantified by ImageJ (I). (J, K) The adhesion function of Sertoli cells was assessed using fluorescence detection(J) after co-culturing mCherry-labeled germ cells with Sertoli cells, and the resulting ratio of germ cells (GCs) to Sertoli cells (SCs) was quantified (K). Scale bars: 100 μm. (L)The mRNA levels of cytokines which influence germ cells in the testis of mice across different groups. (M) Lactate levels in the testis of mice across different groups and normalized to CTL. Statistical analysis between multiple groups was performed by one-way ANOVA. A two-sided p-value < 0.05 was considered to be statistically significant. The level of significance defined as p < 0.05 (*), p < 0.01 (**), p < 0.005 (***), p < 0.001 (****).

Figure 4

Omega-3 alleviates AGE-induced senescence and dysfunction in TM4 cells in vitro. (A) Analysis of TM4 cell survival following treatment with AGEs at a concentration of 200 μg/mL, and the rescuing effects of Omega-3, as determined by the CCK-8 assay. (B-C) The protein expression of RAGE in TM4 cells was detected using Western blotting following treatment with AGEs and an Omega-3 supplement, and quantified by ImageJ (C). (D, F) SA-β-gal activity detection usingβ-galactosidase staining, and statistical analysis of the proportion of positive cells. Scale bar: 50 μm. (E, G) ROS detection using DCFH-DA staining and the fluorescence densities were calculated using ImageJ. Scale bar: 50 μm. (H, I) Analysis of apoptosis using Annexin V-FITC staining by flow cytometry. The late apoptotic rates were calculated and presented in the right panel. (J) The mRNA levels of SASP factors in TM4 cells. (K)Assessment of TM4 cell barrier integrity in vitro. After cell barrier formation, cells were treated and TER detection were used to analyze the integrity of TM4 cell barriers. (L, M) Tight junction protein levels in TM4 cells were examined by western blotting, and quantified by ImageJ (M). (N) Representative images of the formation of cytoskeleton in TM4 cells by F-actin staining immunofluorescence. Scale bar: 20 μm. (O) The mRNA levels of cell adhesion function-related genes in TM4 cells. Statistical analysis between multiple groups was performed by one-way ANOVA. A two-sided p-value < 0.05 was considered to be statistically significant. The level of significance defined as p < 0.05 (*), p < 0.01 (**), p < 0.005(***), p < 0.001 (****).

Figure 5

Omega-3 suppresses the AGE-induced increase in MERCs in Sertoli cells. (A-C) Representative transmission electron microscopy (TEM) images of Mito and ER in Sertoli cells of mice testes from different groups (A). The white asterisks (*) indicate the ER. Quantification of the extent of MERCs (<100 nm) (B) and the Mito-ER proximity in Sertoli cells of mice testis from different groups (n= 12 to 15 mitochondria in 5 fields per condition) (C). (D-F) Representative immunostaining pictures of colocalization between mitochondria and ER in primary Sertoli cells from different groups mice (D). Mitochondria and ER are marked with Mito-tracker and ER-tracker. Scale bar, 10 μm (E) Line intensity profile analysis of Mito-tracker and ER-tracker. Intensity values were measured along a path indicated in lower pictures. The Pearson's correlation between ER-tracker and Mito-tracker was analyzed (F). (G, H) MERCs protein levels in treated TM4 cells were examined by western blotting, and quantified by ImageJ. (I, J) Representative images of proximity ligation assay (PLA) targeting IP3R3-VDAC1 interactions (I) and quantification of the PLA red fluorescent dots (J) in treated TM4 cells (n= 20 to 22 cells in 10 fields per condition). Scale bar: 10 μm. (K, L) Mitochondrial Ca²⁺ detection using Rhod-2 staining (K) and the fluorescence densities were calculated using ImageJ and normalized (L). Statistical analysis between multiple groups was performed by one-way ANOVA. A two-sided p-value < 0.05 was considered to be statistically significant. The level of significance defined as p < 0.05 (*), p < 0.001 (****).

Figure 6

Omega-3 ameliorates AGE-induced ER stress and mitochondrial dysfunction. (A, B) ER stress protein levels in treated TM4 cells were examined by western blotting, and quantified by ImageJ. (C) Protein aggregation was evaluated using PROTEOSTAT^® Protein Aggregation Kit and presented as relative fluorescence units and normalized to CTL level. (D, E) Mitochondrial membrane potential (Δψm) in treated TM4 cells was detected using JC-1 staining (D). Representative images of JC-1 aggregates (red) and JC-1 monomers (green) The ratio of JC-1 aggregates to JC-1 monomers was calculated using ImageJ (E). (F, G) Representative images of MitoSOX (red) fluorescence in treated TM4 cell (F). Scale bar: 100 μm Relative MitoSOX fluorescence was quantitatively analyzed (G). (H) The ATP activity in TM4 cells weas normalized per mg of protein. Statistical analysis between multiple groups was performed by one-way ANOVA. A two-sided p-value < 0.05 was considered to be statistically significant. The level of significance defined as p < 0.05 (*), p < 0.01 (**).

Figure 7

RAGE is a central mediator of AGE-induced Sertoli cell dysfunction. (A-C) Representative immunostaining pictures of colocalization between mitochondria and ER in AGEs treated TM4 with or without the RAGE inhibitor FPS-ZM1(A). Scale bar: 10 μm. Line intensity profile analysis of Mito-tracker and ER-tracker (B). Intensity values were measured along a path indicated in lowest pictures (A). The Pearson's correlation between ER-tracker and Mito-tracker was analyzed (C). (D, E) MERCs protein levels in treated TM4 cells were examined by western blotting. (F, G) Representative immunostaining pictures of mitochondrial Ca²⁺ detection in treated TM4 (F) and the fluorescence densities were calculated using ImageJ and normalized (G). (H, I) SA-β-gal activity detection usingβ-galactosidase staining, and statistical analysis of the proportion of positive cells. Scale bar: 50 μm. (J, K) ROS detection using DCFH-DA staining and the fluorescence densities were calculated using ImageJ. Scale bar: 50 μm. (L) Analysis of TM4 cell survival following treatment with AGEs with or without FPS-ZM1, as determined by the CCK-8 assay. (M) Assessment of TM4 cell barrier integrity in vitro. After cell barrier formation, cells were treated and TER detection were used to analyze the integrity of TM4 cell barriers. (N) Representative images of the formation of cytoskeleton in TM4 cells by F-actin staining immunofluorescence. Scale bar = 20 μm. (O, P) The adhesion function of Sertoli cells was assessed using fluorescence detection (O) after co-culturing mCherry-labeled germ cells with Sertoli cells, and the resulting ratio of germ cells (GCs) to Sertoli cells (SCs) was quantified (P). (Q) The mRNA levels of cytokines which influence germ cells in TM4 cells. (R) The mRNA levels of cell adhesion function-related genes in TM4 cells. Statistical analysis between multiple groups was performed by one-way ANOVA. A two-sided p-value < 0.05 was considered to be statistically significant. The level of significance defined as p < 0.05 (*), p < 0.01 (**), p < 0.005(***), p < 0.001 (****).