Peripheral benzodiazepine receptor/translocator protein global knock-out mice are viable with no effects on steroid hormone biosynthesis

Abstract

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.

Keywords: Adrenal; Cholesterol; Gene Knock-out; Mitochondria; STAR; Steroidogenesis; TSPO.

FIGURE 1.

Generation of TSPO knock-out mice. A, schematic showing genomic Tspo locus in wild type (Tspo^+/+), intact floxed (Tspo^fl/fl), and knock-out (Tspo^−/−) mice. Tspo^fl/fl mice contained exons 2 and 3 flanked with LoxP sites (open arrowheads). Tspo^fl/fl female mice were bred with Ddx4-cre male mice to generate germ cell-specific deletion of TSPO as heterozygotes. These sperm and oocyte-specific knock-out mice were bred to generate Tspo^−/− offspring. Ddx4-cre transgene was subsequently bred out from this colony. B, panel showing genotyping for floxed alleles and cre transgene in Ddx4-cre mice, Tspo^fl/fl mice, Tspo^+/− Ddx4-cre mice, Tspo^−/− Ddx4-cre mice, and Tspo^−/− mice. C, genomic DNA PCR for recombination at the Tspo locus detects deletion of exons 2 and 3 in Tspo^−/− mice (product sizes: Flox-2697 bp, Null-872 bp, and Con-161 bp).

FIGURE 2.

Validation of Tspo^−/− genotype. A, Tspo^fl/fl-ROSA26-tdTomato reporter mice were used to examine cre-mediated recombination induced in offspring from germ cell-specific deletions. Global recombination was clear in R26-tdTom-Tspo^−/− P3 pups. B, TSPO monoclonal antibody specific for peptide corresponding to TSPO exon 4 detected a protein of expected size (18 kDa) in Tspo^fl/fl tissues but not in Tspo^−/− tissues. ACTB and mitochondrial isocitrate dehydrogenase 2 were used as controls. Scale bar, 1 cm. C, representative images of different tissues from an R26-tdTom-Tspo^fl/fl mouse with no tdTomato fluorescence in contrast to tissues from an R26-tdTom-Tspo^−/− mouse showing tdTomato fluorescence (red) indicating recombination. Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm.

FIGURE 3.

TSPO deletion does not affect gonadal steroidogenesis. A, testes sections showing TSPO localization in Leydig and Sertoli cells in Tspo^fl/fl but not in Tspo^−/− testis. Functional morphology of seminiferous tubules was not affected in Tspo^−/− testis. B, ovary sections showing TSPO localization in interstitial cells and surface epithelium, weak in theca and granulosa cells in Tspo^fl/fl ovary; no staining was observed in Tspo^−/− ovary. Functional morphology was not affected in Tspo^−/− ovary. C, base-line plasma testosterone levels were not different between Tspo^fl/fl and Tspo^−/− male mice (n = 24–25/group). D, increase in plasma testosterone levels after human chorionic gonadotropin stimulation was similar between Tspo^fl/fl and Tspo^−/− male mice (n = 6–7/group). E, base-line plasma estradiol levels were significantly higher in Tspo^−/− compared with Tspo^fl/fl mice (p < 0.05), but the difference was modest (n = 12–15/group). F, base-line plasma progesterone levels were not different between Tspo^fl/fl and Tspo^−/− mice (n = 12–15/group). Scale bar, 200 μm.

FIGURE 4.

TSPO deletion has no effect on morphology and lipid deposits in the adrenal cortex. A, adrenal sections from Tspo^fl/fl and Tspo^−/− mice showing TSPO localization in adrenocortical cells with a higher density in the zona glomerulosa; no staining was observed in Tspo^−/− adrenal. No difference in adrenocortical morphology was apparent between two genotypes. B, Oil Red O staining of adrenal glands showed no difference in neutral lipid deposits between Tspo^fl/fl and Tspo^−/− adrenal glands. C, quantification of Oil Red O (ORO) labeling density was similar in both Tspo^fl/fl and Tspo^−/− adrenal cortex (n = 5/group; mean ± S.E.). Scale bar, 200 μm.

FIGURE 5.

TSPO deletion has no effect on adrenal steroid hormone production. A, plasma aldosterone base-line levels were not significantly different between Tspo^fl/fl and Tspo^−/− mice (n = 7/group). B, plasma corticosterone base-line levels were not significantly different between Tspo^fl/fl and Tspo^−/− mice (n = 24–30/group). C, increase in plasma corticosterone in response to ACTH stimulation was similar between Tspo^fl/fl and Tspo^−/− mice (n = 10–14/group). D, representative Western blot showing no compensatory increase in STAR and TSPO protein expression after ACTH stimulation in both Tspo^fl/fl and Tspo^−/− mice. Quantification of STAR (E) and TSPO (F) protein levels also showed no significant increase at 1 h after ACTH stimulation (n = 3). A.U., arbitrary units. Data are represented as mean ± S.E.

FIGURE 6.

TSPO deletion does not alter expression levels of steroidogenic genes and TSPO-interacting proteins in the adrenal glands. Expression levels of steroidogenic genes Star (A), Cyp11a1 (B), and Hsd3b1 (C) were similar between Tspo^fl/fl and Tspo^−/− mice. Tspo2 expression (D) was not detected in both Tspo^fl/fl and Tspo^−/− adrenal glands. Genes encoding TSPO-interacting proteins Vdac1 (E), Vdac2 (F), Vdac3 (G), Ant1 (H), Ant2 (I), Hxk2 (J), Pap7 (K), and Acbp (L) showed similar expression levels between Tspo^fl/fl and Tspo^−/− mice. Tspo expression (M) was not detected in Tspo^−/− adrenal glands (n = 6/group). Data are represented as mean ± S.E. ACBP, acyl-CoA-binding protein; ND, not detected.

FIGURE 7.

Ultrastructure of cells in the adrenal cortex is not affected by TSPO deletion. Transmission electron micrographs showing cell morphology in the zona glomerulosa (A) and zona reticularis (B) of the adrenal cortex. Subcellular organelle morphology, including mitochondria and lipid droplets, is identical between both Tspo^fl/fl and Tspo^−/− mice.

FIGURE 8.

TSPO knockdown in steroidogenic cells does not affect steroid hormone biosynthesis. TSPO knockdown (1 and 2) resulted in substantial decrease in TSPO protein compared with controls (scrambled) in MA-10 (A), Y1 (B), MLTC (D), and R2C cells (E). No base-line TSPO was detected in H295R cells (C). Bt₂cAMP 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 < 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 Bt₂cAMP 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 < 0.05), both with and without Bt₂cAMP treatment.

FIGURE 9.

TSPO expression is not allele-specific. A, TSPO monoclonal antibody detected similar expression levels in Tspo^fl/fl maternally transmitted heterozygous Tspo^+/− and paternally transmitted heterozygous Tspo^+/−; no TSPO was detected in Tspo^−/− adrenal glands. B, there was no significant difference in Tspo mRNA expression levels between Tspo^fl/fl and maternal heterozygous Tspo^+/− adrenal glands; no Tspo mRNA was detected in Tspo^−/− adrenals (n = 4/group; ND*, not detected).