Peripheral Benzodiazepine Receptor/Translocator Protein Global Knock-out Mice Are Viable with No Effects on Steroid Hormone Biosynthesis*♦

Background: Translocator protein (TSPO) has been considered a mitochondrial cholesterol transporter critical for steroid hormone production. TSPO knock-out mice were reported to be embryonic lethal. Results: TSPO knock-out mice are viable with no effects on steroidogenesis. Conclusion: TSPO is not essential for steroidogenesis and is not necessary for sustaining life. Significance: This study rectifies a serious inaccuracy in the current understanding that is critical for treating steroid hormone disorders. Translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is a mitochondrial outer membrane protein implicated as essential for cholesterol import to the inner mitochondrial membrane, the rate-limiting step in steroid hormone biosynthesis. Previous research on TSPO was based entirely on in vitro experiments, and its critical role was reinforced by an early report that claimed TSPO knock-out mice were embryonic lethal. In a previous publication, we examined Leydig cell-specific TSPO conditional knock-out mice that suggested TSPO was not required for testosterone production in vivo. This raised controversy and several questions regarding TSPO function. To examine the definitive role of TSPO in steroidogenesis and embryo development, we generated global TSPO null (Tspo−/−) mice. Contrary to the early report, Tspo−/− mice survived with no apparent phenotypic abnormalities and were fertile. Examination of adrenal and gonadal steroidogenesis showed no defects in Tspo−/− mice. Adrenal transcriptome comparison of gene expression profiles showed that genes involved in steroid hormone biosynthesis (Star, Cyp11a1, and Hsd3b1) were unchanged in Tspo−/− mice. Adrenocortical ultrastructure illustrated no morphological alterations in Tspo−/− mice. In an attempt to correlate our in vivo findings to previously used in vitro models, we also determined that siRNA knockdown or the absence of TSPO in different mouse and human steroidogenic cell lines had no effect on steroidogenesis. These findings directly refute the dogma that TSPO is indispensable for steroid hormone biosynthesis and viability. By amending the current model, this study advances our understanding of steroidogenesis with broad implications in biology and medicine.

Translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is a mitochondrial outer membrane protein implicated as essential for cholesterol import to the inner mitochondrial membrane, the rate-limiting step in steroid hormone biosynthesis. Previous research on TSPO was based entirely on in vitro experiments, and its critical role was reinforced by an early report that claimed TSPO knockout mice were embryonic lethal. In a previous publication, we examined Leydig cell-specific TSPO conditional knock-out mice that suggested TSPO was not required for testosterone production in vivo. This raised controversy and several questions regarding TSPO function. To examine the definitive role of TSPO in steroidogenesis and embryo development, we generated global TSPO null (Tspo ؊/؊ ) mice. Contrary to the early report, Tspo ؊/؊ mice survived with no apparent phenotypic abnormalities and were fertile. Examination of adrenal and gonadal steroidogenesis showed no defects in Tspo ؊/؊ mice. Adrenal transcriptome comparison of gene expression profiles showed that genes involved in steroid hormone biosynthesis (Star, Cyp11a1, and Hsd3b1) were unchanged in Tspo ؊/؊ mice. Adrenocortical ultrastructure illustrated no morphological alterations in Tspo ؊/؊ mice. In an attempt to correlate our in vivo findings to previously used in vitro models, we also determined that siRNA knockdown or the absence of TSPO in different mouse and human steroidogenic cell lines had no effect on steroidogenesis. These findings directly refute the dogma that TSPO is indispensable for steroid hormone biosynthesis and viability. By amending the current model, this study advances our understanding of steroidogenesis with broad implications in biology and medicine.
The peripheral benzodiazepine receptor (PBR) 2 was first identified as a protein with distinct pharmacology and high binding affinity to benzodiazepines in "peripheral" sites, as opposed to "central" sites found in brain tissue (1)(2)(3)(4). The central benzodiazepine receptor is the ␥-aminobutyric acid type A (GABA A ) receptor that has specific function in inhibitory neurotransmission (5,6). However, the function for the PBR has remained a topic of debate for decades (7,8), despite its prominence as a therapeutic target in a variety of diseases/disorders (9 -12).
Genetic sequence comparisons show that the PBR gene is highly conserved from archaea to metazoans (13). In mammals, highly prominent PBR protein expression has been identified in steroidogenic tissues (14 -16). Binding sites for PBR ligands have also been identified in other tissues including heart, brain, adrenal, kidney, salivary gland, platelets, brown adipose tissue, skin, and liver (7). In subcellular fractions, binding sites for the PBR were identified to be present in the outer mitochondrial membrane (OMM) (14,17).
The transport of cholesterol from intracellular stores and the OMM to the inner mitochondrial membrane (IMM) forms the first and rate-limiting step in steroid hormone biosynthesis (18,19). Cholesterol is then converted to the first steroid, pregnenolone, by CYP11A1 (cytochrome P450 side chain cleavage), an enzyme located on the matrix side of the IMM (20,21). The initial link between PBR and steroid hormone biosynthesis emerged by studying the effect of a PBR-binding chemical PK11195 that triggered steroidogenesis in the Y1 mouse adrenal tumor cell line (22). In agreement, targeted disruption of PBR in the R2C rat Leydig tumor cell line inhibited steroidogenesis (23).
The structure of murine PBR showed a five ␣-helix fold, each forming a transmembrane segment (24), and the possibility of a channel-like structure with an interior surface for potential substrate translocation (25). Cholesterol binding to PBR was defined by the identification of a cholesterol recognition amino acid consensus sequence at the C-terminal region (26). These structural features combined with functional considerations led to the modeling of PBR as a membrane-associated cholesterol transport protein (9,27). Based on these findings, to more accurately represent its subcellular role, PBR was renamed as translocator protein (TSPO) (28).
Independent of studies on PBR/TSPO, another mitochondrial protein that is expressed in rapid response to steroidogenic stimuli, the steroidogenic acute regulatory protein (STAR), was identified in the MA-10 Leydig tumor cell line (29). Induced expression of STAR resulted in increased steroid hormone production even in the absence of hormonal stimulation (29). Anchoring STAR in a mitochondrion-affixed TOM20-STAR fusion protein in MA-10 cells resulted in the constitutive production of steroid hormone (30). In humans, STAR mutations cause lipoid congenital adrenal hyperplasia that ranges from an almost complete inability to synthesize steroids (31) to less severe forms that retain partial STAR protein activity (32). Also in support, Star gene-deleted mice showed an almost complete inability to synthesize steroid hormones underscoring its critical role in steroidogenesis (33).
Two key experiments appeared to cement a functional link between STAR and PBR/TSPO. First, inhibition of protein synthesis that blocks STAR production and steroidogenesis (34) did not affect the ability of PK11195 to trigger steroidogenesis (35). Second, knockdown of PBR/TSPO in mitochondrion-affixed TOM20-STAR fusion protein expressing MA-10 cells resulted in a reduction in their steroidogenic capacity (36). Thus, the PBR function was linked to transport of cholesterol from the OMM to the IMM (37).
In a recent study, we questioned this model showing that PBR/TSPO expression is not required for in vivo testicular steroidogenesis (38). This result has indeed raised a lot of questions and controversy regarding the current understanding of the precise PBR/TSPO-mediated functions in steroid hormone production (39). Moving forward, there remained an absolute need to examine specific deficits in TSPO global knock-out mice to gain additional functional understanding of this protein (40).
In a review article published in 1997, it was claimed that efforts to generate a Pbr/Tspo gene-deleted model failed as a result of early embryonic mortality in PBR/TSPO-deficient mice (41). This led to the conclusion that in addition to steroidogenesis, TSPO is involved in basic functions that are critically necessary for embryonic development. However, this review article (41) failed to explain experimental methods covering the recombination strategy used and the exact stage of embryonic mortality; these specific methods/results were never published. Given the current gap in understanding PBR/TSPO function in vivo, we decided to revisit this global knock-out question.
With the primary objective of studying the role of PBR/TSPO in steroidogenesis and other vital functions (if any) that may be critical for embryonic development, we used a germ cell specific knock-out approach to generate Pbr/Tspo global gene-deleted mice. Our results are unexpected and contrary to the prevailing view in that we find PBR/TSPO knock-out mice are viable and fertile with no effects on steroid hormone biosynthesis.
Phenotypic Analysis-Growth rate of Tspo fl/fl and Tspo Ϫ/Ϫ mice were examined from postnatal day 0 to week 5. Litter sizes from homozygous crosses in Tspo fl/fl and Tspo Ϫ/Ϫ mouse colonies were examined in at least eight unique pairings. Recombination was examined in different tissues by both PCR of genomic DNA using specific primers, as validated previously (38), and tdTomato reporter expression indicating cre-mediated recombination, as described previously (38).
Histology-Tissues were processed for hematoxylin and eosin staining as described (38). Immunohistochemistry for PBR/TSPO was performed using a rabbit monoclonal anti-TSPO antibody (Abcam) that specifically recognizes amino acids 156 -169 at the C terminus of exon 4 as described (16,38). For examining neutral lipid deposits, 9-m frozen sections were prepared, and lipid staining was performed using Oil Red O (Matheson Coleman & Bell) in 60% isopropyl alcohol for 30 min. Fluorescent tdTomato was also visualized in 9-m frozen sections in multiple tissues. Images were acquired using either a DFC365FX or an ICC50HD camera (Leica) or a Meta 510 confocal scope (Zeiss). Density of Oil Red O staining density was quantified using ImageJ (44).
Immunoblots-Proteins were separated by SDS-PAGE and immunoblotted using rabbit monoclonal primary antibodies against TSPO (Abcam), isocitrate dehydrogenase 2 (Abcam), and a rabbit polyclonal STAR antibody (45). Each primary antibody was multiplexed with the loading control ACTB (Li-Cor). Simultaneous detection was performed using IRDye 700 and 800 under a laser fluorescence scanner (Li-Cor) and quantified as described previously (38). For immunoblots performed in knockdown experiments, TSPO polyclonal (Cell Signaling) and ACTB (Invitrogen) primary antibodies were used. Specific band intensities in knock-down experiments were calculated by image analysis using Quantity One software (Bio-Rad).
Hormone Assays-Plasma was collected from 8-to 10-weekold Tspo fl/fl and Tspo Ϫ/Ϫ mice. Levels of different steroid hormones were measured using radioimmunoassay as follows: corticosterone, aldosterone, and testosterone (Siemens), estradiol and progesterone (Serono Maia), as described previously (38,46). For adrenal/corticosterone stimulation test, 500 ng/g body weight of adrenocorticotropin (ACTH) fragment 1-24 (Sigma) was administered subcutaneously, and plasma was collected after 1 h. For the testis/testosterone stimulation test, 10 IU of human chorionic gonadotropin (EMD Biosciences) was administered intraperitoneally to male mice, and plasma was collected after 1 h.
Transmission Electron Microscopy-Adrenal glands were fixed in Karnovsky's fixative (2.5% glutaraldehyde, 2% paraformaldehyde in 0.08 M phosphate buffer) overnight. After fixation, tissues were processed for transmission electron microscopy as described previously (12). Stained sections were examined in a Tecnai TM T12 Spirit electron microscope (FEI), at 120 kV. High magnification images were acquired using a high resolution CCD camera Megaview III (Olympus Soft Imaging System) to examine cellular and mitochondrial morphology within regions in the adrenal cortex.
Adrenal Transcriptome Shotgun Sequencing-Adrenal gland total RNA from Tspo fl/fl and Tspo Ϫ/Ϫ mice were extracted, and poly(A) RNA were isolated using oligo(dT) 25 Dynabeads (Invitrogen). Fragmentation and strand-specific RNA-seq library preparation was performed as described (49). Each sample with unique bar codes was pooled to one lane for obtaining short single-reads in a HiSeq 2000 sequencer (Illumina). RNA-seq reads were first aligned to the ribosomal RNA gene database (50) using Bowtie (51), and the mapped reads were discarded. The resulting filtered reads were aligned to the mouse genome (Ensembl) using TopHat (52). Subsequent to alignments, raw counts for each mouse gene were derived and normalized to reads per kb of exon model per million mapped reads. Differentially expressed genes were identified using the DESeq package (53). Raw p values of multiple tests were corrected using false discovery rate (54).

RESULTS
Tspo Ϫ/Ϫ Mice Are Viable and Fertile-The Tspo fl/fl locus allows cre-mediated deletion of exons 2 and 3 of the Tspo gene (Fig. 1A). Exon 1 does not contain any start codons. Removal of exons 2 and 3 eliminates the start codon, and exon 4 does not have any in-frame start codons. Therefore, deletion of exons 2 and 3 will completely eliminate TSPO protein expression (38). Germ cell-specific Ddx4-cre mice were used to induce germ cell Tspo deletion in F1 and global recombination in F2 generations. Litters from all crosses were genotyped, and the Ddx4-cre transgene was subsequently bred out from this colony (Fig. 1B). Using a Tspo fl/fl -R26-tdTom reporter line, we confirmed that cre-mediated recombination occurred in all tissues globally (Fig. 2, A and C). Recombination at the Tspo Ϫ/Ϫ genomic locus was also confirmed by PCR in different tissues (Fig. 1C). The complete absence of TSPO protein expression in different tissues in Tspo Ϫ/Ϫ mice was confirmed by both Western blots (Fig. 2B) and immunohistochemistry (data not shown), using a monoclonal antibody that recognizes protein coded by exon 4. This proved that no partial TSPO peptide was produced in mice deleted for Tspo exons 2 and 3 as shown previously (38). All Tspo Ϫ/Ϫ mice were viable and showed no apparent phenotypic abnormalities. Litter sizes from Tspo Ϫ/Ϫ pair matings were not different from Tspo fl/fl pair litters (Table 1). Growth rate in Tspo Ϫ/Ϫ mice measured up to 5 weeks of age were quite similar to Tspo fl/fl cohorts. However, when analyzed based on sex, Tspo Ϫ/Ϫ female mice showed a very modest (ϳ1 g) but significantly higher body weight than Tspo fl/fl mice at 1-5 weeks of age; males body weights at this same age interval were not different between the two genotypes (data not shown).

Gonadal Steroid Hormone Production Is Not Affected in Tspo
Ϫ/Ϫ Mice-The lack of TSPO did not affect the functional morphology of the testis (Fig. 3A) or the ovary (Fig. 3B) in Tspo Ϫ/Ϫ mice compared with Tspo fl/fl controls. In male mice, examination of plasma testosterone concentration showed that base-line levels were not different between Tspo Ϫ/Ϫ and Tspo fl/fl mice (Fig. 3C). The increase in plasma testosterone in response to human chorionic gonadotropin in vivo was not different between Tspo Ϫ/Ϫ and Tspo fl/fl mice (Fig. 3D). Cauda epididymal sperm counts were not different between Tspo Ϫ/Ϫ and Tspo fl/fl mice (data not shown). In female mice, examination of plasma estradiol and progesterone concentration showed that TSPO deletion did not decrease base-line values (Fig. 3, E and  F). In actuality, Tspo Ϫ/Ϫ mice showed modest but significantly higher estradiol levels compared to Tspo fl/fl cohorts. We did not observe any abnormalities in gonad size, reproductive behavior, and fecundity in Tspo Ϫ/Ϫ mice (data not shown).
Adrenal Steroid Hormone Production Is Not Affected in Tspo Ϫ/Ϫ Mice-The lack of TSPO expression did not affect the functional morphology of the adrenal cortex (Fig. 4A) in Tspo Ϫ/Ϫ mice. Quantitation of neutral lipid staining in the adrenal cortex did not indicate any major steroid transport defects in Tspo Ϫ/Ϫ mice (Fig. 4, B and C). Base-line levels of aldosterone (Fig. 5A) and corticosterone (Fig. 5B), two major hormones produced by the adrenal cortex, were not different between Tspo Ϫ/Ϫ and Tspo fl/fl mice. An increase in plasma corticosterone in response to ACTH in vivo was not different between Tspo Ϫ/Ϫ and Tspo fl/fl mice (Fig. 5C). There was also no evidence for compensatory increase in STAR expression in adrenals of Tspo Ϫ/Ϫ mice compared with Tspo fl/fl mice after ACTH treatment (Fig. 5, D-F).
Loss of TSPO Does Not Affect Steroidogenic Gene Expression in Adrenal Glands-Comparison of gene expression profiles by transcriptome shotgun sequencing of Tspo Ϫ/Ϫ and Tspo fl/fl adrenals revealed only a short list of differentially expressed genes ( Table 2), none of which were directly associated with steroidogenesis. Mining reads representing Tspo mRNA confirmed deletion of exon 2 and exon 3. A majority of the differentially expressed genes was involved in immune activity. Validated expression of genes involved in the steroid hormone biosynthetic pathway, Star, Cyp11a1, and Hsd3b1 (Fig. 6, A-C), and genes encoding TSPO-interacting proteins as follows: Vdac, Ant, Hxk2, Pap-7, and Acbp, were not affected in Tspo Ϫ/Ϫ adrenals (Fig. 6, E-L). Complete dataset is available through NCBI-SRA SRP043599. To evaluate possible functional redundancy, we also examined expression levels for Tspo2, a Tspo paralog (56). We did not detect Tspo2 expression in both Tspo fl/fl and Tspo Ϫ/Ϫ adrenal glands (Fig. 6D).
Ultrastructure of Adrenal Cortex Remains the Same in Tspo Ϫ/Ϫ Mice-Examination of steroidogenic cells of the adrenal cortex using transmission electron microscopy did not show any aberrations in cellular ultrastructure in Tspo Ϫ/Ϫ mice (Fig. 7). Despite its presence in the OMM, TSPO deficiency did not affect mitochondrial morphology. Lipid droplet distribu-  OCTOBER 3, 2014 • VOLUME 289 • NUMBER 40 tion was also identical between regions within adrenal cortex (zona glomerulosa, zona fasciculata, and zona reticularis) in both Tspo fl/fl and Tspo Ϫ/Ϫ mice. TSPO deficiency also did not affect mitochondrial volume in cells, as assessed in Tspo fl/fl and Tspo Ϫ/Ϫ primary fibroblasts that showed no quantitative differences between the two genotypes (data not shown).

PBR/TSPO Is Not Essential for Steroidogenesis
TSPO Knockdown in Steroidogenic Cell Lines Does Not Affect Steroidogenesis-Progesterone production after Bt 2 cAMP treatment by the different murine (MA-10, Y1, and MLTC) and human (H295R) steroidogenic cell lines was not adversely affected by knockdown of TSPO expression (Fig. 8, A-D). TSPO protein expression in murine cell lines decreased by ϳ85, 75, and 82%, respectively; surprisingly, base-line TSPO protein expression was not detectable in human H295R cells (confirmed by extremely low levels of Tspo mRNA (Fig. 8, F and G)). This observation showed that H295R cells are capable of making steroid hormones without TSPO. In all conditions, TSPO knockdown did not have an effect on STAR expression. In the case of MA-10 cells, progesterone production showed a significant increase after Bt2cAMP treatment in TSPO knockdown cells compared with scrambled controls. This is in direct contrast with another study using the same MA-10 cells that showed significantly decreased progesterone production after just 60% knockdown of TSPO protein expression (36). However, such increases were not observed in Y1 and MLTC cells after TSPO knockdown under identical conditions. In R2C cells that abnormally display constitutively high levels of steroidogenesis, TSPO knockdown showed a modest but significant decrease in progesterone production in conditions with and without Bt 2 cAMP treatment compared with scrambled controls (Fig.  8E). Mono-allelic Tspo gene deletion in R2C cells was reported to abolish TSPO expression and inhibit steroidogenesis (23). We find this hard to reconcile, as we do not find any evidence for allelespecific TSPO expression in our in vivo studies (Fig. 9). Moreover, R2C cells are pathologically constitutive in steroid hormone production, the mechanism of regulation of which is not physiologically relevant. Data from TSPO knockdown experiments in three different murine cell lines together with the discovery that TSPO expression is below the detection levels in H295R cells confirm that TSPO is not essential for steroidogenesis in vitro.

DISCUSSION
According to existing literature, TSPO function is absolutely essential for steroid hormone biosynthesis (23), and global Tspo gene deletion results in early embryonic lethality in mice (41). In direct contrast to those observations, our experimental findings disprove these functional arguments and definitively demonstrate that TSPO is not essential for steroidogenesis and that Tspo Ϫ/Ϫ mice are viable and fertile. These concrete results form a key step toward rectifying the prevailing model of steroidogenesis and underscore that the precise role of TSPO in mammalian physiology has yet to be uncovered.
In a previous study, we demonstrated that conditional Tspo gene deletion in testicular Leydig cells did not affect testosterone production (38). At that time, the finding was met with skepticism that Amhr2 cre-mediated recombination may not have complete penetrance in Leydig cells and criticism that it may not be the case for all steroid-synthesizing cells (39). From observations in this study in which Tspo has been deleted globally, it is clear that TSPO is not involved in physiologically relevant steroid hormone biosynthesis. This calls into question the previously suggested functional cooperation between STAR and TSPO for mitochondrial cholesterol import (57).
It could be argued that Tspo Ϫ/Ϫ mice respond to the loss of TSPO with some unknown compensatory mechanism. If this is the case, it is surprising that redundant mechanisms were not evident in earlier studies that claimed an indispensable role for TSPO in the steroidogenic machinery (23). TSPO2, a paralog of TSPO (35% homology) and a likely candidate for functional compensation, is restricted to the adult bone marrow and is not expressed in steroidogenic tissues (56), negating such a possibility. Moreover, there is no experimental evidence that multiple mechanisms are involved in mitochondrial cholesterol import. Based on observations in STAR knock-out mice, it appears unmistakable that there is one rate-limiting mechanism of mitochondrial cholesterol import that is governed by STAR (33). Therefore, a model for TSPO in mitochondrial cholesterol transport is highly unlikely.
Given these results, it is unclear how the global TSPO knockout reported in an earlier study by Papadopoulos et al. (41) could be embryonic lethal. As exact experimental details for that study were not published, we can only speculate that an error occurred in genome manipulation during this previous attempt. Similarly, a study from this same research group reported that more than 70% TSPO knockdown in cell lines resulted in cell death (58). This is certainly not the case in both our in vivo and in vitro experimental observations and indicates that experimental conditions used in these earlier studies may have not been optimal. This conclusion is supported by another recent study that independently generated conditional liverand heart-specific Tspo Ϫ/Ϫ mice and did not report any incidence of cell death after abolishing TSPO expression; TSPO was found not to be involved in mitochondrial permeability transition and cell death (59).
At base-line physiology, gene expression differences between Tspo fl/fl and Tspo Ϫ/Ϫ adrenals were surprisingly few. A majority of these genes are involved in immune modulation as follows: Tmem178 is a negative regulator of inflammatory cytokine production (60); Prrc2a is a gene within the major histocompatibility complex class III region and is involved in the inflammatory process (61); Osgin1 encodes an oxidative stress response protein that regulates inflammation (62); Zbtb7b is a regulator of T cell-mediated tumor-induced immunity (63); Pydc4 is part of a PYHIN domain family of proteins involved in innate immunity (64); and Trim12a is a putative antiviral gene (65). Two genes were involved in vesicular transport and lipid sequestration as follows: Epn1 is an endocytic adaptor involved in clathrin-mediated endocytosis (66), and Abca2 encodes an endoly- . Bt 2 cAMP treatment resulted in a similar increase in STAR protein expression in MA-10, Y1, MLTC, and H295R cells and higher progesterone production. TSPO knockdown MA-10 cells showed significantly higher progesterone production (p Ͻ 0.05) compared with scrambled controls. Progesterone production in TSPO knockdown Y1 and MLTC cells was similar to the scrambled controls. H295R cells did not show TSPO expression but still produced progesterone upon Bt 2 cAMP treatment. Careful re-examination confirmed that H295R cells do not express both TSPO protein and mRNA (F and G). Abnormal R2C cells constitutively expressed high STAR protein levels and constantly produced high levels of progesterone. TSPO knockdown in R2C resulted in a modest but statistically significant decrease in progesterone production (p Ͻ 0.05), both with and without Bt 2 cAMP treatment. sosomal protein with a role in intracellular lipid trafficking (67). Three genes expressed were of unknown function: Ankrd52, Abhd1, and Atxn2l. The remainders were two noncoding RNA genes, two predicted genes, and three pseudogenes. Careful consideration of these differentially expressed genes and the phenotype observed in Tspo Ϫ/Ϫ mice indicates that TSPO is not essential for sustaining physiological function. TSPO binding chemicals have been shown to increase steroid hormone production in isolated mitochondria from steroidogenic cells (35), MA-10 cells (37), and even live rats (68). Given our current findings, we can offer two possible explanations for these pharmacological effects. With ligand binding, TSPO may undergo a conformational change (as suggested previously (24,69)), resulting in the release of bound cholesterol that subsequently becomes available to mitochondrial transport systems and steroidogenesis. Alternatively, the effect of pharmacological agents could be due to drug-membrane interactions resulting in TSPO-independent biological effects (70). It has also been discovered that TSPO ligands like PK11195 can insert themselves into lipid bilayers affecting membrane properties (71), something that can modulate cholesterol availability to the steroidogenic machinery and also the nature of TSPOligand interactions. Nevertheless, as TSPO is considered a therapeutic target for numerous inflammatory conditions (9 -12), these properties of TSPO ligands may be important to assess specificity and side effects.
The sexual disparity in body weight differences in that only female Tspo Ϫ/Ϫ mice showed subtle but higher growth weights than their Tspo fl/fl cohorts suggests a hormone-dependent metabolic mechanism (72). But the overall lack of phenotype in Tspo Ϫ/Ϫ mice appears to suggest that TSPO function could be more relevant only in pathological disease states. We have now observed Tspo Ϫ/Ϫ mice for up to 16 months of age with no apparent abnormalities/problems. Comparison of gene expression profiles after TSPO deletion also suggests potential changes to immune system function associated with TSPO deletion. We intend to investigate this further in future studies to gain insight into TSPO function.
In summary, this study provides irrefutable evidence that PBR/TSPO is not essential for steroid hormone biosynthesis and that Tspo Ϫ/Ϫ mice are viable and fertile. These findings form a critical step toward understanding TSPO function and will help rebuild a defined model for steroid hormone biosynthesis. In addition, this understanding also forces reevaluation of interpretations made for TSPO function across multiple fields, including neuroscience, immunology, and oncology.