High fat diet causes distinct aberrations in the testicular proteome

Abstract

Diet has important effects on normal physiology and the potential deleterious effects of high fat diets and obesity on male reproductive health are being increasingly described. We conducted a histological review of the effects of chronic high fat (HF) diet (using a mouse model fed a 45% fat diet for 21 weeks) with a discovery proteomic study to assess for changes in the abundance of proteins in the testis. Mice on a HF diet became obese and developed glucose intolerance. Using mass spectrometry, we identify 102 proteins affected in the testis of obese mice. These included structural proteins important for the blood testis barrier (filamin A, FLNA), proteins involved in oxidative stress responses (spermatogenesis associated 20, SPATA-20) and lipid homoeostasis (sterol regulatory element-binding protein 2, SREBP2 and apolipoprotein A1, APOA1). In addition, an important regulator protein paraspeckle component 1, PSPC-1, which interacts with the androgen receptor was significantly downregulated. Proteomic data was validated using both Western blotting and immunostaining which confirmed and localised protein expression in both mouse and human testis using biopsy specimens. This study focused mainly on the abnormalities that occurred at the protein level and as a result, we have identified several candidate proteins and conducted pathway analysis around the effects of HF diet on the testis providing novel insights not previously described. Some of the identified targets could be targeted therapeutically and future work is directed in this area.

Fig. 1. Effects of the HF diet on body weight and glycaemic control.

a Experimental set up and use of tissues. b Body weight. Mice were weighed weekly prior to and after receiving the HF (n = 9) or NC diets (n = 7). c Effect of HF diet on fasting glucose when mice were tested at 8 weeks of age when all were on the same diet and then repeated at 27 weeks of age (16 weeks after commencing HF diet) or ongoing NC diet. d Effect of HF diet on glucose and (e) glucose tolerance. Statistical analysis performed using 2-way ANOVA ***p < 0.001 comparing HF to control. Abbreviations Oil red O (ORO), Haematoxylin & Eosin (H&E), Immunofluorescence (IF), intraperitoneal glucose tolerance test (ipGTT).

Fig. 2. Histological alterations in response to HF diet in the mouse testes.

Representative histological images of H&E stained testicular tissues from mice fed either a HF diet (I,iii) as compared to control mice fed a NC diet (ii, iv) at the end of study (aged 32 weeks). Narrowing of seminiferous tubules is highlighted (black arrow).

Fig. 3. Summary of proteomic data after multivariate analysis with depiction of differentially expressed proteins in the testes between HF and NC conditions.

a Multivariate analysis of proteomic data demonstrating 3D scores plot from unsupervised principal components analysis (PCA) showing separation between the testicular tissues from mice fed a HF diet compared to NC. b Hierarchal cluster heatmap based on protein expression between HFD vs. Chow mice and the contribution to the separation of phenotypes by unique protein IDs to either HF or NC diets. Data is presented as technical triplicates for each mouse sample analysed (n = 3). Each lane represents a technical triplicate (Created in Metaboanalyst v3.0). c Venn diagram representation of proteins in the testis between the HF and NC conditions. 1012 proteins were found uniquely in NC mouse testes whilst 920 were uniquely in HF mice. 3028 proteins were expressed in both conditions of which 102 proteins were significantly altered in HF testes. The Log2 FC fold change for these 102 proteins ranged between −2.34 and 1.39 (p < 0.05). d Direction of the differentially expressed statistically significant proteins in HF condition (n = 102 proteins).

Fig. 4. Top IPA pathways deregulated in the testes in HF diet versus NC conditions.

a Top Ingenuity pathways associated with altered proteins in the testes from mice fed a HF diet as compared to NC mice. Bars indicate the likelihood [−log (P-value)] that the specific pathway is affected by dietary fat. Protein IDs generated after LC-MS/MS analysis of the global testicular proteome in HF and NC fed mice were put through IPA to generate a total number of curated proteins relevant to that pathway identified in the whole testis. The p value represents the pathway significance to the tissue. Testis specific pathways related to Germ cell-Sertoli cell junction signalling; Germ cell:Germ cell signalling are highlighted in orange. b Top pathway downregulated in testis from HF conditions: caveolar mediated endocytosis signalling, green depicts downregulated proteins in the pathway including FILAMIN A, FILAMIN B, ITGA11 (alpha integrin subunit 11), PTRF/CAV1 (caveolae associated protein).

Fig. 5. Western blot validation studies of 6 key protein targets differentially expressed in the testes from mice on HF diet versus NC conditions.

a HF diet decreases the protein expression of FLNA, AR and PSPC-1, SREBP2, APOA-I with an increased in SPATA20 expression. b Western blots of all proteins extracted from testicular tissues from 3 representative mice and (c) semi-quantitative protein expression using band densitometry analysis using image J. Values are average +/−SEM (n = 3 per group). *p < 0.05, **p < 0.01, ***p < 0.001 and compared using by two tailed unpaired t test.

Fig. 6. Immunofluorescence imaging comparing altered protein targets (FLNA, PSPC- 1, SREBP2) between representative testes from mice fed NC and HF diet and expression in human testis.

Representative immunofluorescence images comparing altered protein targets (FLNA, PSPC-1, SREBP2) between representative testes from (a) mice fed NC and HF Mouse testis. All images at presented at ×63, scale bar represents 50 μm. b Representative immunofluorescence staining of selected protein targets in human testicular biopsies (from patients with complete spermatogenesis (iv–vi) and Sertoli cell only (vii–ix) compared to images from Human Protein Atlas (i–iii) and mouse testis (x–xii). All images at ×20 and scale bar represents 50 μm.