Mitochondrial dynamics, Leydig cell function, and age‐related testosterone deficiency

The mitochondrial translocator protein (18 kDa; TSPO) is a high‐affinity cholesterol‐binding protein that is an integral component of the cholesterol trafficking scaffold responsible for determining the rate of cholesterol import into the mitochondria for steroid biosynthesis. Previous studies have shown that TSPO declines in aging Leydig cells (LCs) and that its decline is associated with depressed circulating testosterone levels in aging rats. However, TSPO's role in the mechanistic decline in LC function is not fully understood. To address the role of TSPO depletion in LC function, we first examined mitochondrial quality in Tspo knockout mouse tumor MA‐10 nG1 LCs compared to wild‐type MA‐10 cells. Tspo deletion caused a disruption in mitochondrial function and membrane dynamics. Increasing mitochondrial fusion via treatment with the mitochondrial fusion promoter M1 or by optic atrophy 1 (OPA1) overexpression resulted in the restoration of mitochondrial function and mitochondrial morphology as well as in steroid formation in TSPO‐depleted nG1 LCs. LCs isolated from aged rats form less testosterone than LCs isolated from young rats. Treatment of aging LCs with M1 improved mitochondrial function and increased androgen formation, suggesting that aging LC dysfunction may stem from compromised mitochondrial dynamics caused by the age‐dependent LC TSPO decline. These results, taken together, suggest that maintaining or enhancing mitochondrial fusion may provide therapeutic strategies to maintain or restore testosterone levels with aging.


| INTRODUCTION
Testicular Leydig cells (LCs) are the main sites of testosterone production in men and contribute to the maintenance of circulating testosterone levels. 1 Testosterone biosynthesis is essential for the development, function, and maintenance of the male reproductive system. 1 At the approximate age of 30 in humans, testosterone levels begin to decline at a rate of 0.4%-2% annually and can lead to testosterone deficiency, known as hypogonadism. [1][2][3] Due to the importance of testosterone in biological systems, hypogonadism is accompanied by a number of conditions, including decreased muscle mass, fat mass accumulation, mood changes, fatigue, metabolic syndrome, and others. 4 The first step in LC testosterone production involves the metabolism of cholesterol by the inner mitochondrial membrane enzyme cytochrome P450 side chain cleavage (CYP11A1). The transfer of cholesterol from intracellular reserves into mitochondria, the rate-limiting step in steroidogenesis, is essential for this process. 5 Possessing a high affinity for cholesterol, the 18-kDa translocator protein (TSPO) is an essential outer mitochondrial membrane protein that is abundant in steroidogenic cells. 6 TSPO contains a binding domain known as the cholesterol recognition amino acid consensus motif with a strong affinity for cholesterol. 6 Coordinating with a variety of cytosolic and mitochondrial membrane proteins which form the Steroidogenic InteracTomE (SITE), 7 TSPO participates in the targeting of cholesterol to CYP11A1. [7][8][9] Although TSPO expression declines in aging LCs, 10 it is not yet known how this decline may be linked to the development of hypogonadism. Furthermore, the mechanisms mediating the transport of cholesterol from the outer to the inner mitochondrial membrane are not fully understood.
Lipids are transported through subcellular membranes via three mechanisms: vesicular trafficking, facilitated diffusion with soluble lipid transfer proteins, and diffusion across membrane contact sites. The vesicular trafficking network precludes mitochondria vesicle transport, 11 and therefore cholesterol trafficking to the mitochondria is likely mediated by transfer proteins at mitochondrial membrane contact sites. 12,13 Contact sites have long been identified as a possible route for mitochondrial cholesterol transport. [14][15][16] Contact sites between mitochondrial membranes are involved in molecular transport and are formed by proteins localized to the outer and inner mitochondrial membranes. 17 One such mitochondrial membrane pore protein, the voltage-dependent anion channel (VDAC1), provides passage for a variety of molecules into the mitochondria 18 and forms a complex with TSPO. 8 Interestingly, TSPO depletion in LCs disrupts the VDAC/tubulin interaction, suggesting that TSPO influences mitochondrial pore stability. 13 Mitochondria are highly dynamic organelles constantly undergoing fusion and fission throughout their life cycle. 19 These alterations in mitochondrial structure impact a number of functions, including cellular bioenergetics, 20 mitochondrial degradation, 21 and oxidative stress. 22 Moreover, mitochondrial fusion is needed for steroidogenesis and declines with aging. 23 Mitochondrial fusion is mediated through the mitochondrial contact site and cristae-organizing system (MICOS) complex. 24 Interactions among MICOS proteins in the inner membrane space bring together the inner and outer mitochondrial membranes. 20 One such protein, the mitochondrial membrane GTPase optic atrophy 1 (OPA1), narrows cristae junctions and increases contact site formation. 25 Opa1 mRNA is spliced to produce long and short isoforms which promote limited activity on their own, but together their co-expression improves fusion. 26 Opa1 deletion results in fewer and more fragmented cristae among mitochondria and a reduction in complex IV subunits, disrupting mitochondrial respiration. 27 OPA1 was thought to be involved in steroidogenesis, but its depletion in the tumorigenic MA-10 mouse LC line failed to disrupt hormone biosynthesis and brought into question its involvement. 8 However, redundancies in the MICOS system and regulation of mitochondrial dynamics remain. 20 Nevertheless, TSPO expression may influence mitochondrial fusion, as its insertion into the T-cell Jurkat cell line led to numerous increases in MICOS-related genes. 28 Furthermore, Tspo deletion depolarizes mitochondrial membrane potential in MA-10 LCs, 13 which destabilizes mRNA cleavage of the Opa1 isoform producing long OPA1. 29 Along with TSPO decline, imbalanced mitochondrial fusion dynamics have been observed in aged and dysfunctional LCs. 30 TSPO's elimination in MA-10 LCs causes aberrant morphology in mitochondrial contact sites, suggesting that TSPO influences mitochondrial integrity. 13 However, studies examining mitochondrial dynamics in aged and dysfunctional LCs are limited.
TSPO is an integral protein in health whose activation can regulate cell death and promote protective and restorative responses. [31][32][33] Despite numerous studies of TSPO's role in mitochondrial function, 34 no studies have revealed why its decline in LCs leads to mitochondrial dysfunction or how this relates to reduced steroid formation. To study the potential role of TSPO in the regulation of mitochondrial function, we utilized a TSPO knockout (KO) nG1 MA-10 sub-cell line, 13 isolated LCs from rats containing a Tspo deletion, 35 and LCs isolated from aging rats with reduced TSPO levels. 10 In an effort to restore LC mitochondrial function and given TSPO's association with bioenergetics in LCs, we promoted mitochondrial fusion and thus contact site formation using two approaches: overexpression of Opa1 and treatment with the mitochondrial fusion promoter, 4-Chloro-2-(1-(2-(2,4,6-trichlorophenyl) hydrazono) ethyl) phenol (M1).

| Animals
Young (2 months old), middle-aged (6.5 months old), and old (12-16.5 months old) Sprague-Dawley rats (n = 4 for each group) that carry Tspo deletion mutation (SD-Tspo em5Vpl; RAT5) were generated using zinc finger nuclease technology, as we previously reported. 35 These and control rats were maintained according to protocols approved by the Institutional Animal Care and Use Committee of the University of Southern California (Protocol # 20791). Rats were killed by decapitation. Trunk blood was collected, and plasma was separated by centrifugation at 2000 g for 15 min, stored at −80°C, and used for determination of circulating testosterone levels.

| Primary LC isolation
LCs were isolated from young (2 months old), middleaged (6.5 months old), and old (12-16.5 months old) control and Tspo mutant rats using isosmotic continuous Percoll (Gibco Inc.) gradients generated by centrifugation, as previously described with minor modifications. 36 LCs were isolated from two rats from each age group and the experiments were repeated three times unless noted otherwise. Briefly, testes were perfused, decapsulated, and dissociated using 0.25 mg/ml collagenase shaken at 80 cycles/min at 34°C for 15 min. Following dissociation, the supernatant was collected and centrifuged at 800 g for 20 min. Resuspended pellets were placed into a Percoll density gradient and centrifuged at 14 000 rpm for 45 min at 4°C. The LC-enriched fraction was isolated and placed atop a BSA density gradient and centrifuged at 50 g for 10 min, yielding an 85% pure LC solution. LC purity was determined by 3β-hydroxysteroid dehydrogenase chemical reaction, as described previously. 36 In brief, LC fractions were incubated for 30 min at 37°C with the substrate dehydroepiandrosterone (100 g/ml; Sigma-Aldrich, St. Louis, MO, USA) in 0.07 M phosphate buffer (pH 7.2) containing 1 mg/ml nicotinamide, 6 mg/ml g-NAD and 1.5 mg/ml nitro blue tetrazolium (Sigma-Aldrich).

| Immunoblot analysis
Protein was extracted using RIPA buffer supplemented with protease inhibitors. Protein concentration was measured using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed using 1 μg/μl of purified protein on a 4%-20% Tris-glycine gradient gel (Bio-Rad, Hercules, CA, USA). Protein bands were electro-transferred to a polyvinylidene fluoride membrane and blocked with 5% BSA for 30 min.
The OPA1 mouse monoclonal antibody clone 1E8-1D9 that reacts with mouse, rat, and human protein was from ThermoFisher Scientific (product #MA5-16149). TSPO protein expression was assessed using an affinity-purified rabbit anti-peptide antibody raised against the mouse TSPO C-terminal sequence as previously described. 39 This antiserum detects TSPO in human, mouse and rat tissues, and cells. 40 Membranes were incubated with primary antibody overnight at 4°C at 1:1000 dilution and secondary antibody at 1:5000 for 1 h at room temperature. Membranes were then quenched with Radiance Peroxide and Radiance Plus (Azure Biosystems, Dublin, CA, USA) and subsequently imaged with an Azure c600 system (Azure Biosystems). Membranes were then stripped with Restore Western Blot Stripping Buffer (ThermoFisher Scientific) and incubated with a housekeeping antibody for normalization. Anti-sera against beta-actin (Danvers, MA, USA), alpha-tubulin (Sigma-Aldrich), and GAPDH (Proteintech, Rosemont, IL, USA) were used as controls.

| Measurement of steroid hormones
MA-10 wild-type and nG1 cells (1 × 10 4 ) were plated on 96-well plates for 24 h. Media was removed, wells were washed with phosphate-buffered saline, and media treatments were added. Cells were treated with 50 ng/ml human chorionic gonadotropin (hCG; National Hormone and Peptide Program, Harbor-UCLA Medical Center Torrance, CA, USA) or control media and incubated for 2 h at 37°C. Media were collected for steroid measurement using the Progesterone ELISA Kit (Cayman Chemical, Ann Arbor, MI, USA), and cells were lysed with 0.1 N sodium hydroxide for subsequent protein measurement using the Bradford Protein Assay (Avantor, Radnor, PA, USA).
Similarly, isolated primary LCs (1 × 10 5 ) from wild-type young and aging rats and the Tspo mutant RAT5 were collected in microfuge tubes, placed in media, and shaken at 80 cycles/min at 34°C for 2 h. Testosterone measurement was performed using the Testosterone ELISA Kit (Cayman Chemical). Plasma testosterone levels were measured using the same ELISA kit.

| Measurement of cellular respiratory function
Cultured cells (1 × 10 4 ) and isolated primary LCs (3 × 10 4 ) were plated onto Seahorse XF Cell Culture Microplates overnight (Agilent Technologies, Santa Clara, CA, USA). Cell media were replaced with Agilent Seahorse XF DMEM Medium supplemented with 1 mM glucose, 1 mM pyruvate, and 2 mM glutamine and incubated at 37°C in a non-CO 2 incubator. Cells were evaluated using the Seahorse XF Cell Mito Stress Test Kit or the Seahorse XF Real-Time ATP Rate Assay Kit according to the manufacturer's specifications. Briefly, working solutions of Oligomycin (2.5 μM), FCCP (2.0 μM), and Rot/ AA (0.5 μM) were prepared and loaded into a sensor cartridge that had been hydrated in Seahorse XF Calibrant at 37°C in a non-CO 2 incubator overnight. The assay was performed using the Seahorse XFe96 Analyzer with templates designed in Wave 2.6.1.

| Mitochondrial imaging
Cells were imaged by transmission electron microscopy (TEM) at the USC Core Center of Excellence in Nano Imaging. Cells were primarily fixed using 2.5% glutaraldehyde, 2% paraformaldehyde, 0.1 M HEPES, and 0.115 M Sucrose. After washing with 0.1 M cacodylate, cells were F I G U R E 1 TSPO deletion decreased mitochondrial function in MA-10 Leydig cells. (A) Mitochondrial stress test in MA-10 and nG1 Leydig cells with oxygen consumption quantified using Agilent Wave. (B) ATP rate assay in MA-10 and nG1 Leydig cells. (C) Basal and hormone-stimulated (50 ng/ml hCG) progesterone formation after two-hour treatment in MA-10 and nG1 Leydig cells measured via ELISA. Data are presented as mean ± SEM (n = 3). *p < .05 **p < .01 ***p < .001 by two-tailed student's t-test. Antimycin A, Antimycin A1b; FCCP, trifluoromethoxy carbonylcyanide phenylhydrazone; OCR, oxygen consumption rate.
placed in a secondary fixative containing 1% osmium tetroxide. Cells were then stained with uranyl acetate and dehydrated with a series of 30%-100% of EtOH washes. The dehydrated cells were transitioned to a microfuge tube using propylene oxide and infiltrated using increasing concentrations of polybed 812 epoxy resin. The cellcontaining-block was sectioned using the Leica EM UC6 Ultramicrotome (Leica Biosystems, Nussloch, Germany) and examined on the FEI Talos F200C G2 Biological Transmission Electron Microscope (ThermoFisher Scientific).

| Immunofluorescence and confocal microscopy
Cultured cells were fixed in 4% paraformaldehyde in phosphate-buffered saline for 10 min at room temperature. After washing with phosphate-buffered saline, cells were incubated in 0.1% triton for 10 min at room temperature. After washing and blocking with 5% horse serum, cells were incubated with anti-TSPO and anti-OPA1 antibodies at 1:400 dilution, or anti-TOMM20 antibodies (Abcam; product #ab56783) at 1:4000 overnight at 4°C. After washing, samples were incubated with appropriate Alexa Flour dyes 1:400 dilution at room temperature for 30 min, and subsequently stained with DAPI. Digital confocal images were captured with a Zeiss laser scanning 700 series confocal microscope wavelength 400-750 nm (Zeiss, Jena, Germany) at the USC School of Pharmacy Translational Research Laboratory. Images were processed and quantified using the ImageJ software (National Institute of Health, United States). Mean fluorescence intensity was measured and represented as a percentage difference from the MA-10 control.

| Statistical analysis
Data from experiments performed in triplicate are expressed as the mean ± standard error of the mean. GraphPad Prism (v.7; GraphPad Software, San Diego, CA, USA) was used for graphical presentation and statistical analysis. Analysis was performed using a student's t-test or ANOVA with multiple comparisons where appropriate. Results were considered statistically significant at p < .05.

| Tspo deletion decreased mitochondrial function in MA-10 LCs
Previous studies have shown that Tspo deletion in MA-10 LCs alters mitochondrial integrity and the ability of the cells to produce progesterone. A major objective of the present study was to determine the quantitative relationship between TSPO and mitochondrial function. Cells were grown in Seahorse plates to assess mitochondrial function in TSPO deficient nG1 LCs compared to wild-type MA-10 LCs. As seen in Figure 1A, basal cellular respiration, mitochondrial proton leak, maximal cellular respiration, ATP production, and spare respiratory capacity were significantly decreased in nG1 cells. The nG1 oxygen consumption rate was decreased over the MA-10 cells. The alterations seen in our results represent declining regulation of bioenergetic machinery. Mitochondrial ATP is essential to steroidogenic capacity and supports steroidogenesis. 41 Evaluating the ATP production rate using Seahorse 96-well plates revealed significant decreases in both the mitochondrial and glycolytic ATP production rates, and therefore a significant decline in total ATP production ( Figure 1B). To determine if the alterations in ATP production rate alter steroidogenesis, we conducted an enzyme-linked immunosorbent assay (ELISA) to measure steroid production. As seen in Figure 1C, although both wild-type and nG1 MA-10 cells responded to hCG treatment, both basal and hormone-stimulated progesterone formation showed significant declines in nG1 cells.

| Increased mitochondrial fusion restores bioenergetics in TSPOdeficient LCs
The mitochondria of LCs are highly active in fusion and fission, the interplay of which is integral to mitochondrial turnover and the transport of molecules into and from the mitochondria. 23 To investigate the relationship between mitochondrial fusion and function in LCs, we induced mitochondrial fusion by treatment of MA-10 cells with a cellpermeable phenylhydrazone, M1, which has been shown to promote mitochondrial tubular network formation, increase OPA1 expression, and increase ATP levels. 37,38 As seen in Figure 2A, treatment with M1 resulted in increased mitochondrial functions and increased steroid biosynthesis. The oxygen consumption rate and hormone production of the M1-treated nG1 cells reflected that of the control MA-10 cells, but MA-10 cells treated with M1 showed decreased mitochondrial function (Figure 2A). To follow up on this, mitochondria were stained with TOMM20 to assess the mitochondrial population in M1treated cells. Confocal images showed an excessive buildup of mitochondria in M1-treated MA-10 cells, whereas nG1 cells treated with M1 showed mitochondrial populations like that of control MA-10 cells ( Figure 2B). Given that M1 increases the levels of the mitochondrial protein OPA1, cells were transfected with Opa1. Opa1 transfection increased mitochondrial function and hormone-stimulated steroidogenesis in nG1 ( Figure 2B).

| Characterization of Opa1 transfection and M1 treatment in MA-10 and nG1 LCs
Imbalanced fusion dynamics may compromise the integrity of the steroidogenic protein scaffold essential for cholesterol transport. 30 Therefore, we assessed how the expression of proteins in the steroidogenic scaffold is altered in response to increased mitochondrial fusion. OPA1 overexpression was visualized in Opa1-transfected LCs. Immunoblots revealed overexpression of OPA1 in transfected MA-10 and nG1 cells, as well as significant increases in Opa1 and Tspo gene expression levels in transfected MA-10 cells ( Figure 3A). Immunoblot analysis of steroidogenic-related proteins revealed increases in CYP11A1 and VDAC, but not in the bioenergetic proteins adrenodoxin (ADX) or ATP complex V beta subunit (ATPB) (Figure 3B), suggesting that bioenergetic proteins are not upregulated despite greater ATP production ( Figure 2). M1 treatment resulted in increases in opa1 and tspo gene expression in MA-10 cells ( Figure 3C). TSPO gene expression increased slightly in M1-treated MA-10 cells, but other proteins involved in steroid formation did not ( Figure 3D). To further address the relationship between TSPO levels and the treatment of MA-10 cells with M1 or transfection with Opa1, immunochemical analyses were conducted to relate OPA1 and TSPO expressions. Increases were seen in OPA1 and TSPO levels after treatment with M1 or transfection with Opa1 ( Figure 4A). nG1 samples also showed increased fluorescence for TSPO in M1-treated and Opa1-transfected samples ( Figure 4B), similar to the results found with MA-10 cells.

| Mitochondrial fusion improved mitochondrial morphology in TSPO-deficient LCs
With the knowledge that OPA1 regulates aspects of mitochondrial cristae dynamics, we hypothesized that structural deviations seen in nG1 cells might improve by upregulating fusion. Dysfunctional mitochondria have aberrant cristae structures are replaced via mitochondrial biogenesis. 20,42 Using transmission electron microscopy imaging to visualize mitochondrial morphology, we found that nG1 mitochondrial morphology was restored after M1 treatment or Opa1 transfection ( Figure 5). MA-10 cells presented healthy mitochondria with the expected cristae morphology (black arrows), while dysfunctional mitochondria were prevalent in nG1 cells (red arrowheads). Fusion upregulation via M1 and Opa1 overexpression reduced the number of dysfunctional mitochondria in nG1 cells. Cristae prevalence and structure (yellow doublesided arrows) were also restored in fusion-upregulated nG1 cells. The restored mitochondrial and cristae structure indicates the greater respiratory capacity and bioenergetic function, consistent with the findings in Figure 2.

| M1 treatment improves bioenergetics and steroid formation in aged rat LCs
To evaluate whether the upregulation of mitochondrial fusion enhances steroidogenic output and mitochondrial function, LCs were isolated from 2-month-old and 1-year-old rats and treated with M1. Basal respiration, maximal respiration, and ATP production were significantly higher in the LCs of the 2-month-old rats. LCs isolated from 1-year-old animals had significant increases in maximal respiration and spare capacity. Basal respiration and ATP production were increased at this age, albeit not significantly ( Figure 6A). Serum testosterone and testosterone produced from isolated LCs showed declines over the rat lifespan ( Figure 6B). LCs from rats aged 1 year produced significantly increased levels of testosterone after M1 treatment (Figure 6C), suggesting a role for mitochondrial fusion in the maintenance of steroidogenic function. Tspo mutant RAT5 animals produced less testosterone in rats of 2, 6.5, and 16.5 years of age when compared to the WT. Testosterone levels were greater in M1-treated 16.5-month-old WT and Tspo-mutant animals ( Figure 6D).

| DISCUSSION
Alterations in cholesterol transport and TSPO expression may play critical roles in age-related testosterone decline. Aging LCs become TSPO deficient compared to young cells, 10,43 and this could disrupt the translocation of cholesterol into the mitochondria 10,44,45 ; the rate-limiting step in steroidogenesis. 46,47 TSPO is integrally involved in the translocation of cholesterol into the mitochondria for steroid biosynthesis and its depletion in LCs leads to functional declines. 9,13,35,[48][49][50] Given the impact of TSPO depletion on steroid production and mitochondrial integrity, 10,13,35,44,51 we tested the effect of increasing mitochondrial fusion on steroid biosynthesis by upregulating OPA1 via drug treatment and Opa1 overexpression. Our rationale for these approaches came from previous studies showing that OPA1 expression reduces mitochondrial 52 dysfunction and regulates cristae remodeling. 53 Therefore, we anticipated upregulation would improve LC function. We discovered that OPA1 upregulation indeed restored steroid biosynthesis and LC function in TSPO-depleted cells. In these studies, we used both transient transfection and pharmacological means to induce mitochondrial fusion. The two approaches resulted in consistent findings, increasing our confidence that enhanced mitochondrial fusion ameliorates dysfunction in TSPO-deficient LCs. Consistent with these findings, previous studies reported that the activation of TSPO with a TSPO drug ligand led to the stabilization of mitochondrial architecture during inflammation stress in cells of the colon, and to reduced cell death. 54 It should be noted that there have been studies based on the knockdown Opa1 that have suggested that Opa1 may not be critical for steroidogenesis. 8 While OPA1 may not be indispensable for steroid formation, it might be the case that compensatory mechanisms may be involved in mitochondrial remodeling for cholesterol transport into the mitochondria. This is supported by the finding that there are a large number of steroidogenic proteins in the steroidogenic interactome (SITE), and numerous MICOS complex proteins. 8,25 Interestingly, the OPA1 protein expression fold change increase in MA-10 transfected cells was higher than in nG1 transfected cells, suggesting that TSPO stabilizes mitochondrial fusion dynamics. This is consistent with previous findings, as nG1's disrupted membrane potential 13 likely results in the destabilization of Opa1 mRNA cleavage, limiting fusion capacity. 29 The loss of mitochondrial membrane potential destabilizes mRNA cleavage which produces the long isoform of OPA1. 29 The deteriorated membrane potential in nG1 may disrupt the fusion potential in M1-treated nG1 cells, as coexpression of the long and short OPA1 isoforms enhance fusion. 29 Moreover, our results identify a relationship between OPA1 and TSPO expression. Immunoblot, qPCR, and immunocytochemistry staining studies revealed increases in TSPO expression after M1 treatment or Opa1 transfection, suggesting that OPA1 may regulate TSPO expression. However, it may be that TSPO regulates OPA1, as our data showed depressed OPA1/Opa1 expression in nG1 samples. Opa1 mRNA cleavage is compromised when the mitochondrial membrane potential is disrupted, which may explain the dynamics between these two proteins seen in our results given TSPO's influence on membrane potential. Puzzlingly, there was an increase in TSPO fluorescence in nG1 samples after Opa1 transfection. OPA1 overexpression may be influencing transcription factors regulating TSPO expression, amplifying residual expression. MA-10 cells have an aberrant chromosome number. Previous studies demonstrated that the extinction of one allele of the Tspo gene in the constitutively steroidproducing R2C LC line abolished TSPO expression. 55 It is possible that OPA1 may drive Tspo expression from the remaining alleles.
The role of OPA1 in disease progression is well known. Variations in Opa1 expression have been implicated in a number of diseases including dominant optic atrophy, deafness, and spinocerebellar degeneration. 56 These disruptions manifest reductions in oxygen consumption, cell viability, and ATP synthase assembly. 57 Given OPA1's role in mitochondrial function, we were not surprised that increasing OPA1 expression improved function in TSPO-deficient LCs. However, we were intrigued to find that increased OPA1 levels improved steroid hormone formation in LCs. It may be the case that increasing OPA1 levels improve mitochondrial contact site presence and cristae integrity, increasing the efficiency of cholesterol binding and transport. Similarly, Opa1 overexpression increased TSPO and VDAC levels in MA-10 LCs, suggesting that mitochondrial fusion may regulate the expression of SITE proteins. Given the cholesterol-binding properties of TSPO, these increases may enhance cholesterol translocation into the mitochondria. The cholesterol recognition amino acid consensus is oriented such that the presence of a binding interface between TSPO and VDAC is likely. 58 Previously, modeling VDAC structures revealed a likely shift among backbone amides in the presence of cholesterol 59 and reasonable binding orientations for cholesterol in specific VDAC sites have also been identified. 60 Taken together, our results indicate a relationship between mitochondrial fusion and steroidogenic capacity.
The formation of mitochondrial cristae, the main sites of oxidative phosphorylation, is regulated by OPA1. 61 OPA1 modulates oxidative phosphorylation by oligomerizing and narrowing cristae junctions, leading to ATP synthase dimerization. 53 Morphologically, the curvature is induced which physically narrows the distance between the electron transport chain and enhances ATP synthesis efficiency. 62 Electron microscopy revealed disruptions to mitochondrial morphology and declining cristae formation in TSPO-deficient LCs. Given morphological and proton gradient irregularities previously identified in TSPO-deficient LCs, 13 we anticipated a decline in ATP synthesis. As expected, ATP production was significantly decreased in TSPO KO LCs, which was restored with OPA1 overexpression, along with steroid formation. While declines in respiration are seen in quiescent cells, alterations in proton leak impact the coupling efficiency and reactive oxygen species production in cells. 63 Much of the proton leak is attributed to the adenine nucleotide translocase (ANT), which collaborates with oxidative phosphorylation machinery to regulate mitochondrial respiration. 63,64 Given that steroid synthesis by CYP11A1's side chain cleavage of cholesterol requires electrons from complex III and IV of the electron transport chain, 65 cristae improvements may enhance electron availability for steroid biosynthesis. Inhibition of mitochondrial ATP reduces all steps in the steroidogenic pathway and produces reduced steroid output. 52 Narrowing of cristae junctions enhances mitochondrial ATP formation and likely provides electrons for side chain cleavage. It is possible that TSPO depletion's impact on ATP synthesis may disrupt the flow of electrons for cholesterol side chain cleavage.
A confounding result of the present studies is that there was declining mitochondrial respiration in the M1-treated MA-10 cells. One possible explanation could involve the physiological role of mitochondrial fusion in cells exposed to selective stresses. Hyperfusion of mitochondria is mediated through OPA1 and creates an excessive interconnected tubular network in response to a selective stressor. 26,66 Indeed, confocal imaging revealed excessive and connected mitochondrial populations in the MA-10 cells treated with M1, a characteristic commonly observed in hyperfusion. 66 In addition to increased mitochondrial fusion, treatment may be increasing stressor signaling. Interestingly, this trend was not observed in M1-treated isolated primary rat LCs. Primary LCs had improved mitochondrial function after the M1 treatment. Moreover, testosterone biosynthesis was enhanced in isolated LCs from WT and Tspo-mutant RAT5 animals aged 16.5 months. This may suggest that TSPO may not be directly involved in mitochondrial fusion, but it may play a role in the integrity of the contact site complex. Nevertheless, increased fusion resulted in enhanced steroid hormone formation. This could be related to the inherent differences between the tumorigenic cell line and primary LCs. A noteworthy difference between these models is that MA-10 LCs derive a greater proportion of their ATP from glycolysis. As such, MA-10 LCs employ a larger partition of glycolytic ATP for cholesterol transport when compared to primary cells. 52 Furthermore, primary cells utilize mitochondrial ATP for ER enzymatic reactions that are absent in the tumorigenic LCs. 52,67 These inherent differences demonstrate the need for thorough investigation using various models.
A sound therapeutic strategy to combat primary hypogonadism requires a comprehensive understanding of the translocation of cholesterol into the mitochondria. TSPO decline in aging LCs is correlated with cellular dysfunction and suboptimal steroid biosynthesis production. This decline is likely the result of insufficient cholesterol trafficking into the mitochondria. Findings indicating that mitochondrial fusion is essential for steroidogenesis 23 support the idea that mitochondrial contact sites may act as a conduit for steroidogenesis. 16 Our model (Figure 7) suggests that TSPO depletion leads to declining mitochondrial cristae integrity which causes depressions in mitochondrial function and steroidogenesis in LCs. The declining function may be ameliorated by increased fusion, as contact sites likely play a role in regulating steroid hormone formation in addition to mitochondrial function. Encouragingly, increased fusion in isolated primary LCs from aged rats improved testosterone production, revealing a potentially novel strategy to approach hypogonadism.