Chronic stress inhibits testosterone synthesis in Leydig cells through mitochondrial damage via Atp5a1

Abstract Stress is one of the leading causes of male infertility, but its exact function in testosterone synthesis has scarcely been reported. We found that adult male rats show a decrease in bodyweight, genital index and serum testosterone level after continual chronic stress for 21 days. Two‐dimensional gel electrophoresis (2‐DE) and MALDI‐TOF‐MS analysis identified 10 differentially expressed proteins in stressed rats compared with controls. A strong protein interaction network was found to be centred on Atp5a1 among these proteins. Atp5a1 expression significantly decreased in Leydig cells after chronic stress. Transfection of Atp5a1 siRNAs decreased StAR, CYP11A1, and 17β‐HSD expression by damaging the structure of mitochondria in TM3 cells. This study confirmed that chronic stress plays an important role in testosterone synthesis by regulating Atp5a1 expression in Leydig cells.

hypothalamus; follicle-stimulating hormone and luteinizing hormone are inhibited by blocking of gonadotropin-releasing hormone, thereby inhibiting the secretion of testosterone, decreasing sperm number and vitality and damaging male reproductive function. 4 Previous scientific evidence has suggested that psychological stress affects spermatogenesis, mainly through the regulation of secretion of nerve and endocrine hormones. Testosterone is an important hormone that maintains male reproductive function, and 95% of the testosterone in mammals is secreted by Leydig cells in the testes.
StAR in the mitochondria of Leydig cells regulates the transportation of cholesterol from the outer to the inner mitochondrial membrane; the cholesterol is then transferred to CYP17A1 and converted to pregnenolone. A portion of pregnenolone passes through 3β-HSD in the endoplasmic reticulum of the sliding surface. The remainder is directly converted into dehydroepiandrosterone by CYP17A1, then metabolized into rostenedione by 17β-HSD and finally directly converted into testosterone. 5 The steroid hormone synthase and proteins including StAR, CYP17A1, 3β-HSD and 17β-HSD, which are required for testosterone synthesis, are mainly distributed in the mitochondria. In recent years, mitochondria have increasingly been recognized for their important roles in fertility. Testosterone has been reported to regulate the expression of mitochondrial genes and alleviate oxidative damage, 6 and mitochondrial antioxidants protect steroidogenesis, on the basis of the expression of testosterone and its related steroid synthase. 7 Lipoprotein metabolism induced by obesity disrupts the electron transport chain and ultimately leads to reduced mitochondrial membranes and inhibits testosterone biosynthesis in Leydig cells. 8 In addition, acetamiprid drugs have a similar effect. 9 Thus, mitochondrial dysfunction has been speculated to be closely associated with testosterone synthesis and male reproduction. However, the regulation of cholesterol transport in Leydig cells under stress requires further study. This study aimed to identify changes in gene expression patterns in the testis induced by chronic stress. We used proteomics techniques and then screened for differential indicators involved in male fertility to explore whether stress induces mitochondrial damage and regulates abnormal testosterone synthesis.
Proteomics technology has been widely used in research on a variety of clinical diseases, and is a mature technical method for studying the testis proteome in mammals. Proteomic studies have shown that human sperm contains 20 differentially expressed proteins, thus providing a basis for identifying infertility-related proteins through proteomics technology. 10 In addition, 2-DE and mass spectrometry analysis have identified the roles of prostatic acid phosphatase and prostate-specific antigen in sperm damage and normal conditions in humans, 11 as well as other proteins involved in human reproduction. 12 In summary, the potential mechanisms of molecules involved in male reproductive dysfunction can be explored through testicular proteomics analysis. Herein, 2-DE combined with MALDI-TOF-MS analysis was used to identify changes in the testicular proteome under chronic stress, to reveal the protein expression associated with testosterone synthesis (Figure 1). Subsequently, TM3 cells were used to determine whether the differential indicators might be involved in the testosterone synthesis pathway. This study provides a reliable theoretical basis on the effects of human psychological pressure on male reproductive function.

| Animals and model preparation
Forty-eight healthy adult male SD rats (280 ± 20 g) were purchased from the Animal Center of Xi'an Jiaotong University (Medical Experimental Animal Centre of Shaanxi Province, China), and randomly divided into stressed and control groups (n = 24 per group, four rats per cage) after a 7-day adaptation period. The rats were kept at a constant temperature of 22 ± 2°C and a humidity of 50%, and were given free access to drinking water and food. All experiments were performed in accordance with the relevant guidelines and regulations, and all animal procedures were approved by the Animal Ethics Committee of Xi'an Jiaotong University (No. XJTULAC2019-1272). Chronic stress for 21 consecutive days including food deprivation (24 h), fear sound F I G U R E 1 All experimental procedures performed in this study stimulation (9:00-12:00 a.m. and 15:00-18:00 p.m.), water deprivation (24 h), moist litter (24 h), ice water swimming (5 min, 1-minute interval, 3 times), empty bottle stimulation (24 h) and day and night inversion (change every 12 h) in turns. All treatment types are listed in Table S1. The control group received no treatments.

| Sample collection
Rats were sacrificed after anaesthesia, and then, the testis and epididymis on both sides were separated on ice. Collected tissues were immediately frozen in liquid nitrogen and then transferred to −80°C for storage. The weights of the body, testis, and epididymis were recorded at 3 days, 7 days, 14 days, and 21 days.

| ELISA
The concentration of testosterone in the serum was detected with double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kits (Elisa Biotech). Standards and samples were added to the wells, the target antibody and HRP-conjugated secondary antibody were then added according to the instructions. The data were measured at 450 nm with a microtitre plate reader (FLUO star Omega, BMG LABTECH GmbH).
After isoelectric focusing, samples were separated on a 12% SDS polyacrylamide gel and sealed with 0.5% agar. When the bromophenol blue indicator reached the bottom, electrophoresis was stopped, and the gel was stained. Finally, we performed a transmission scan at 300 DPI. Differential protein spots were analysed, and in gel trypsin digestion was performed for further identification.

| Mass spectrometric analysis and protein identification
Differential spots in 2-DE gels were manually excised and washed, and then 50 mM NH 4 CO 3 /CH 3 CN (1:1) was added for decolourization.
After vacuum-drying, 10 mM DTT was added to each sample and reacted at 56°C for 1 h, and samples were then digested with trypsin and Instrument parameters and analysis were as previously described. 14

| Immunohistochemistry
Paraffin sections (5 µm) of the testis were deparaffinized and rehydrated with xylene and gradient ethanol (100%, 95%, 90% and 80%), and endogenous peroxidase activity was eliminated. Antibodies were incubated at 4°C overnight after antigen retrieval, and the secondary antibody was incubated the next day. Immunolabelling was revealed F I G U R E 2 Chronic stress damages male reproductive organs and perturbs hormone levels. A, The concentration of testosterone (T) in the serum in male rats (n = 6 per group, **p < 0.01). B, C, Weight index comparison of the testis and epididymis between the stressed and control group (*p < 0.05) with 3,3´-diaminobenzidine (ZSGB-BIO). Sections were counterstained with haematoxylin, dehydrated in graded ethanol (80%, 90%, 95% and 100%), rendered transparent with xylene twice and sealed with neutral gum for further analysis.

| siRNA synthesis and Transfection
Sequences of Atp5a1 siRNA and scrambled siRNA (Table S2) were pre-designed and synthesized by the GenePharma Corporation.

JetPRIME Transfection Reagent (Polyplus Transfection) was used
for the transfection of TM3 cells.

| qRT-PCR analysis
Total RNA was extracted from TM3 cells and then quantified with a NanoDrop Microvolume Spectrophotometer (Thermo). qRT-

PCR was performed with an FTC-3000P Real-Time Quantitative
System (Funglyn Biotech), with PrimeScript RT Reagent and an SYBR Premix Kits (Genestar). All primers used are presented in Table S3.

| Transmission electron microscopy
Samples were fixed with glutaraldehyde for 48 h, then dehydrated with an acetone gradient and embedded and cut into semi-thin sections (0.1 mm). After staining with uranyl acetate and lead citrate, samples were finally observed with a H-7650 electron microscope (Hitachi).

| Western blot analysis
Cells were lysed in RIPA buffer and then centrifuged (800 g, 10 min, at 4°C). Protein quantification was detected with a BCA Protein Assay Kit (Pioneer Biotechnology). Equal amounts of protein (20 μg) were separated with 10% SDS-PAGE and subsequently transferred to PVDF membranes (Millipore). Membranes were blocked 1 h at room temperature, then incubated with primary antibodies overnight at 4°C (Table S4). The corresponding secondary antibodies were incubated for 2 h at room temperature after TBST washing. Immunoblots were visualized with ECL (Pierce) for chemiluminescence detection.

| Data analysis
A Bio-Rad GS800 scanner was used for two-dimensional electrophoresis gel scanning. PDQuest professional software was used for qualitative and quantitative mapping analysis for the differential spots from testis tissue proteins in the stressed group and control group.
Protein spots that differed by at least twofold, according to software analysis, were considered differential protein spots and subjected to digestion by trypsin followed by MALDI-TOF-MS mass spectrometry. Mass spectrometry analysis was performed with the GPS Explorer software database to determine differential proteinrelated information.
qRT-PCR data were analysed with the 2 −ΔΔCt method, and the target gene expression was normalized to GAPDH expression. The positive intensity of immunohistochemistry was scored with Hscore in Image J 2.0 (NIH). All data are expressed as mean ±SD and were analysed in GraphPad Prism version 5.01 (GraphPad Software). P < 0.05 was considered statistically significant (*).

| Chronic stress decreases testosterone levels and damages reproductive organs
Male rats showed raised hair, nervousness, irritability and mutual fighting on the first day under stress conditions, and showed reduced activity, irregular hair, sluggishness and unresponsiveness after 3 days of stress. Serum testosterone levels significantly decreased after 21 days of stress ( Figure 2A). The epididymal index in the stressed group was significantly lower than that in the control group on the 21st day, whereas there was no difference in the testicular index ( Figure 2B,C). These results suggested the effects of chronic stress on testosterone levels and reproductive organ damage.

| Chronic stress affects testis protein expression patterns
To assess the effects of chronic stress on the expression of proteins in the testis, we constructed 2-DE maps with high resolution for the testis at day 21, which showed that 375.5 ± 15.5 and 407.5 ± 17.5 protein spots (mean ± SD) differentiated the control and stressed groups ( Figure 3A).
The protein profiles were subjected to PDQuest gel analysis, and the results indicated in 25 differentially expressed protein spots ( Figure 3B).
The 13 spots with the most significant difference were separated from 2-DE gels, and 10 proteins were finally identified by MALDI-TOF-MS ( Figure 3C, Table 1, Figure S1). Among these proteins, Akap4, Atp5a1 and Eno1 were downregulated in the stressed group compared with the control group, whereas Pkm2, Prss2, Ywhaz, Acsm2 and Myl9 were upregulated. Got1 and Uqcrc2 were expressed in the stressed group but F I G U R E 3 Chronic stress induces differential protein expression in 2-DE maps of the testis. A, High-resolution 2-DE maps of proteins extracted from the testis in rats in the stressed and control groups on day 21 (n = 6 per group). B, The replicable protein spots were analysed in PDQuest. A total of 25 differentially expressed spots were screened on the basis of fold differences in gray value relative to the control group. C, Subsequently, 13 of the 25 spots were selected as the most significant in the statistical analysis from the 2-DE gels. Each spot differentially present between the stressed and control groups is listed. Gels were stained with Coomassie brilliant blue, and the original maps are provided in Figure S1 Spots no. TA B L E 1 Mass spectrometry information of differential expression proteins not the control group. The spectra and molecular weights of these differentially expressed proteins are provided in Table 1.  Figure 4B). In addition, the immunohistochemistry analysis of 21-day testicular tissue showed that the Atp5a1-positive region was mainly distributed in Leydig cells, and its expression was lower in the stressed group than the control group ( Figure 4C). Overall, these results indicated that the decrease in Atp5a1 was associated with the synthesis of testosterone in Leydig cells.

| Atp5a1 affects testosterone synthesis via mitochondrial dysfunction
Atp5a1 has been reported to be associated with male sperm motility. Because the immunohistochemistry results revealed that different expression levels of Atp5a1 were mainly distributed in Leydig cells, we speculated that Atp5a1 might be associated with the secre- into the cytoplasm significantly increased ( Figure 5F). These results indicated that Atp5a1 restrains the synthesis of testosterone in Leydig cells and may also regulate apoptosis pathway ( Figure 5G).

| DISCUSS ION
Psychological stress is an important factor for the development of male reproductive dysfunction. 3 Stress events increase the psychological burden and accelerate illness, including male infertility. 15  Calpain-1, thus contributing to diabetic cardiomyopathy. 24 Through interaction with apoptosis regulators, it also maintains mitochondrial membrane potential and regulates skeletal muscle, thereby contributing to exercise endurance in mice. 25 However, few studies have found that the downregulation of Atp5a1 is involved in mitochondrial function and sperm quality. 26 In this study, proteomic analysis  37 Ywhaz has also been reported to be involved in spermatogenesis through regulating PP1γ2. 38,39 Ywhaz and its binding partners play important role in protein-protein interactions during spermatogenesis. 40 Ywhaz was found in the testis in male rats under stress by the proteomic analysis in this study, thus providing a research breakthrough in testicular F I G U R E 5 Atp5a1 is involved in the regulation of testosterone synthesis. A, Transfection of TM3 cells with Atp5a1 siRNAs decreased the target gene expression level relative to that of a control gene (GAPDH), as assessed by qRT-PCR. B, Mitochondria (black arrows) in the TM3 cells were observed by transmission electron microscopy (bar = 2 µm, partially enlarged, 500 nm). The rate of abnormal mitochondria was recorded in at least five random sections. The bar chart shows the length (C), width (D) and rate of abnormal mitochondria (E) in TM3 cells. An asterisk represents statistically significant differences (*p < 0.05, **p < 0.01). F, Expression levels of StAR, CYP11A1, 17β-HSD and Cyt C in cytoplasm were evaluated by western blot analysis. G, Diagram showing how Atp5a1 transfection in TM3 cells downregulates Atp5a1 expression in mitochondria and promotes mitochondrial damage, thus inhibiting the expression of testosterone synthesis genes reproduction. Uqcrc2 and Eno1 are significantly correlated with fertility. 28 Uqcrc2 is a component of the biquinol-cytochrome reductase complex, which is associated with spermatogenesis, 41 and has been shown to be a biomarker for clinical varicocele and asthenozoospermic testicular cancer patients associated infertility. 42 The results of our study showed that Uqcrc2 plays a negative role in spermatogenesis, 43 but the specific pathway requires further study.
Atp5a1 and Uqcrc2 are mitochondria-related genes. Eno1 and Pkm2 are glycolytic enzymes, 44 and Pkm2 accumulation in the cell nucleus has been shown to be decreased by Ywhaz inhibition, thus suggesting a close relationship between them. 45

| CON CLUS IONS
This study showed that psychological stress affects body weight and reproductive organs in adult male rats and alters protein expression patterns in the rat testis. More importantly, decreased Atp5a1 involved in the testosterone synthesis pathway in Leydig cells and is potentially closely associated with damage to male reproductive organs. Strengthening mental health and improving lifestyle to reduce the negative effects from stress are important to maintain normal male fertility. However, the incidence of male infertility has continued to rise in recent years. The main underlying reasons are complex, and the pathogenic mechanism of male reproductive dysfunction requires in-depth exploration in future studies.

CO N FLI C T S O F I NTE R E S T
All authors declare no conflicts of interest regarding this study.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available.