Type 1 diabetes impairs the activity of rat testicular somatic and germ cells through NRF2/NLRP3 pathway-mediated oxidative stress

Abstract

Background: It is well known that metabolic disorders, including type 1 diabetes (T1D), are often associated with reduced male fertility, mainly increasing oxidative stress and impairing the hypothalamus-pituitary-testis (HPT) axis, with consequently altered spermatogenesis and reduced sperm parameters. Herein, using a rat model of T1D obtained by treatment with streptozotocin (STZ), we analyzed several parameters of testicular activity.

Methods: A total of 10 adult male Wistar rats were divided into two groups of five: control and T1D, obtained with a single intraperitoneal injection of STZ. After 3 months, the rats were anesthetized and sacrificed; one testis was stored at -80°C for biochemical analysis, and the other was fixed for histological and immunofluorescence analysis.

Results: The data confirmed that T1D induced oxidative stress and, consequently, alterations in both testicular somatic and germ cells. This aspect was highlighted by enhanced apoptosis, altered steroidogenesis and Leydig cell maturity, and impaired spermatogenesis. In addition, the blood-testis barrier integrity was compromised, as shown by the reduced levels of structural proteins (N-cadherin, ZO-1, occludin, connexin 43, and VANGL2) and the phosphorylation status of regulative kinases (Src and FAK). Mechanistically, the dysregulation of the SIRT1/NRF2/MAPKs signaling pathways was proven, particularly the reduced nuclear translocation of NRF2, affecting its ability to induce the transcription of genes encoding for antioxidant enzymes. Finally, the stimulation of testicular inflammation and pyroptosis was also confirmed, as highlighted by the increased levels of some markers, such as NF-κB and NLRP3.

Conclusion: The combined data allowed us to confirm that T1D has detrimental effects on rat testicular activity. Moreover, a better comprehension of the molecular mechanisms underlying the association between metabolic disorders and male fertility could help to identify novel targets to prevent and treat fertility disorders related to T1D.

Keywords: INSL3; RXFP2; SIRT1; apoptosis; blood-testis barrier; inflammasome; spermatogenesis; steroidogenesis.

Figure 1

Analysis of oxidative stress parameters in control and T1D rat testis. (A) Testicular TBARS levels and (B) SOD and CAT enzymatic activities. (C) WB analysis of testicular SOD, CAT, and 4-HNE protein levels. (D–F) Histograms showing SOD, CAT, and 4-HNE relative protein levels, quantified using ImageJ and normalized to β-actin. (G) Testicular 4-HNE (green) immunolocalization. The slides were counterstained with PNA (red) and DAPI-fluorescent nuclear staining (blue). The images were captured at ×20 (scale bars = 20 µm) magnification and ×40 (scale bars = 10 µm) for the insets. Arrows, SPG; arrowheads, SPC; dotted arrows, SPT; triangle, SPZ tails; striped arrows, SC; asterisks, LC. (H) Histogram showing the quantification of 4-HNE fluorescence signal intensity. All values are expressed as means ± SEM from five animals in each group. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2

Analysis of apoptotic rate in control and T1D rat testis. (A) WB analysis of testicular p53, Bax, Bcl-2, cytochrome-C, and caspase-3. (B–E) Histograms showing the p53, Bax/Bcl-2 ratio, cytochrome-C, and caspase-3 relative protein levels quantified using ImageJ and normalized to β-actin. (F) Determination of apoptotic cells through the detection of TUNEL-positive cells (green). The slides were counterstained with PNA lectin (red) and with DAPI-fluorescent nuclear staining (blue). The images were captured at ×20 (scale bars = 20 µm) magnification. Arrows, SPG; arrowheads, SPC; asterisks, LC. (G) Histogram showing the percentage of TUNEL-positive cells. All the values are expressed as means ± SEM from five animals in each group. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

Analysis of LC activity in control and T1D rat testis. (A) Testicular Testosterone levels and (B) WB analysis of StAR, 3β-HSD, CYP17A1, and CYP19A1 protein levels. (C–F) Histograms showing StAR, 3β-HSD, CYP17A1, and CYP19A1 relative protein levels quantified using ImageJ and normalized to β-actin. (G) Testicular StAR (red) and 3β-HSD (green) immunolocalization. The slides were counterstained with DAPI-fluorescent nuclear staining (blue). The images were captured at ×20 (scale bars = 20 µm) magnification and ×40 (scale bars = 10 µm) for the insets. Asterisks, LC. (H, I) Histogram showing the quantification of StAR and 3β-HSD fluorescence signal intensity, respectively. (J) WB analysis of INSL3 and RXFP2 protein levels. (K, L) Histograms showing INSL3 and RXFP2 relative protein levels quantified using ImageJ and normalized to β-actin. All values are expressed as means ± SEM from five animals in each group. *p < 0.05; **p < 0.01.

Figure 4

Analysis of spermatogenesis in control and T1D rat testis. (A) WB analysis of testicular PCNA, p-H3, H3, and SYCP3. (B–D) Histograms showing PCNA the p-H3/H3 ratio and SYCP3 relative protein levels quantified using ImageJ and normalized to β-actin. (E) Testicular PCNA (green) and SYCP3 (red) immunolocalization. The slides were counterstained with DAPI-fluorescent nuclear staining (blue). The images were captured at ×20 magnification (scale bars = 20 µm). Arrows, SPG; arrowheads, SPC. (F, G) Histograms showing the percentage of PCNA- and SYCP3-positive cells, respectively. All the values are expressed as means ± SEM from five animals in each group. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

Analysis of blood–testis barrier markers in control and T1D rat testis. (A) WB analysis of testicular N-CAD, OCN, ZO-1, CX43, VANGL2, p-Src, Src, p-FAK-Y397, p-FAK-Y407, and FAK in the testes of T1D animals. (B–I) Histograms showing N-CAD, OCN, ZO-1, CX43, and VANGL2 relative protein levels and p-Src/Src, p-FAK-Y39/FAK, and p-FAK-Y407/FAK ratios quantified using ImageJ and normalized to β-actin. All the values are expressed as means ± SEM from five animals in each group. *p < 0.05; **p < 0.01.

Figure 6

Immunofluorescent analysis of N-CAD, ZO-1, and OCN in control and T1D rat testis. (A) Testicular N-CAD (green) immunolocalization. The slides were counterstained with PNA lectin (red) and DAPI-fluorescent nuclear staining (blue). (C) Testicular ZO-1 (green) and OCN (red) immunolocalization. (E) Testicular ZO-1 (green) and β-actin (red) immunolocalization. All the slides were counterstained with DAPI-fluorescent nuclear staining (blue). All the images were captured at ×20 (scale bars = 20 µm) magnification and ×40 (scale bars = 10 µm) for the insets. Dotted arrows, SPT; striped arrows, SC. (B, D, F) Histograms showing the quantification of N-CAD, OCN, and ZO-1 fluorescence signal intensity, respectively. All the values are expressed as means ± SEM from five animals in each group. *p < 0.05.

Figure 7

Immunofluorescent analysis of CX43 and VANGL2 in control and T1D rat testis. (A) Testicular CX43 (green) immunolocalization; the slides were counterstained with PNA lectin. (C) Testicular VANGL2 (green) and α-tubulin (red) immunolocalization. All the slides were counterstained with DAPI-fluorescent nuclear staining (blue). All the images were captured at ×20 (scale bars = 20 µm) magnification and ×40 (scale bars = 10 µm) for the insets. Arrows, SPG; arrowheads, SPC; dotted arrows, SPT; striped arrows, SC. (B, D) Histograms showing the quantification of CX43 and VANGL2 fluorescence signal intensity, respectively. All the values are expressed as means ± SEM from five animals in each group. **p < 0.01; ***p < 0.001.

Figure 8

Analysis of SIRT1/NRF2/MAPKs pathways in control and T1D rat testis. (A) WB analysis of testicular SIRT1, FOXO1, KEAP1, HO-1, p-p38, p38, p-JNK, and JNK. (B–G) Histograms showing SIRT1, FOXO1, KEAP1, and HO-1 relative protein levels and p-p38/p38, and p-JNK/JNK ratios quantified using ImageJ and normalized to β-actin. (H) WB analysis of testicular NRF2 protein levels in cytoplasmic (Cyt) and nuclear (Nuc) fractions. (I) Histograms showing NRF2 relative protein levels quantified using ImageJ and normalized to GAPDH or PCNA. (J) Testicular NRF2 (green) and SIRT1 (red) immunolocalization. The slides were counterstained with DAPI-fluorescent nuclear staining (blue). The images were captured at ×20 (scale bars = 20 µm) magnification and ×40 (scale bars = 10 µm) for the insets. Arrows, SPG; arrowheads, SPC; dotted arrows, SPT. (K, L) Histograms showing the quantification of NRF2 and SIRT1 fluorescence signal intensity, respectively. All the values are expressed as means ± SEM from five animals in each group. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 9

Analysis of pyroptosis in control and T1D rat testis. (A) WB analysis of testicular NF-κB, IL-6, NLRP3, and caspase-1. (B–E) Histograms showing NF-κB, IL-6, NLRP3, and caspase-1 relative protein levels quantified using ImageJ and normalized to β-actin. (F) Testicular NF-κB (green) immunolocalization. (H) Testicular NLRP3 (green) immunolocalization. All the slides were counterstained with PNA lectin (red) and with DAPI-fluorescent nuclear staining (blue). The images were captured at ×20 (scale bars = 20 µm) magnification and ×40 (scale bars = 10 µm) for the insets. Arrows, SPG; arrowheads, SPC; dotted arrows, SPT. (G, I) Histograms showing the quantification of NF-κB and NLRP3 fluorescence signal intensity, respectively. All the values are expressed as means ± SEM from five animals in each group. **p < 0.01; ***p < 0.001.