Translocator protein/peripheral benzodiazepine receptor is not required for steroid hormone biosynthesis

Abstract

Molecular events that regulate cellular biosynthesis of steroid hormones have been a topic of intense research for more than half a century. It has been established that transport of cholesterol into the mitochondria forms the rate-limiting step in steroid hormone production. In current models, both the steroidogenic acute regulatory protein (StAR) and the translocator protein (TSPO) have been implicated to have a concerted and indispensable effort in this cholesterol transport. Deletion of StAR in mice resulted in a critical failure of steroid hormone production, but deletion of TSPO in mice was found to be embryonic lethal. As a result, the role of TSPO in cholesterol transport has been established only using pharmacologic and genetic tools in vitro. To allow us to explore in more detail the function of TSPO in cell type-specific experimental manipulations in vivo, we generated mice carrying TSPO floxed alleles (TSPOfl/fl). In this study we made conditional knockout mice (TSPOcΔ/Δ) with TSPO deletion in testicular Leydig cells by crossing with an anti-Mullerian hormone receptor type II cre/+ mouse line. Genetic ablation of TSPO in steroidogenic Leydig cells in mice did not affect testosterone production, gametogenesis, and reproduction. Expression of StAR, cytochrome P450 side chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase type I, and TSPO2 in TSPOcΔ/Δ testis was unaffected. These results challenge the prevailing dogma that claims an essential role for TSPO in steroid hormone biosynthesis and force reexamination of functional interpretations made for this protein. This is the first study examining conditional TSPO gene deletion in mice. The results show that TSPO function is not essential for steroid hormone biosynthesis.

Figure 1.

Generation of TSPO conditional knockout mice. A, Schematic showing recombination stages. Exons 2 and 3 were flanked with LoxP sites (open arrowheads), using a vector that also carries a neomycin resistance (neoR) selectable marker flanked by Frt sites (vertical double-headed black arrows). Correctly recombined embryonic stem cell (ESC) clones were used to generate mice through blastocyst injections. Germline transmitting TSPO-targeted mice were crossed with ubiquitous Flpe-expressing mice to remove neoR cassette. TSPOfl/fl mice were bred with Amhr2cre/+ knock-in mice, resulting in the deletion of exons 2 and 3 in target cells. Genotyping primers are indicated as P1, P2, and P3. B, Long-range PCR for selecting ES cell clones. Six correctly targeted clones were identified (N, negative control; P, positive control). C, Specific DNA primers (P1, P2, and P3) were used to genotype and identify the floxed and wild-type alleles in TSPO-targeted mice.

Figure 2.

Amhr2cre/+-mediated gene deletion in Leydig and Sertoli cells. Testis from ROSA26-tdTomato (R26-tdTom) reporter mice showing controls with no recombination and specific recombination in Leydig (L) and Sertoli (S) cells with Amhr2cre/+ expression (R26-tdTom-Amhr2cre/+ mice). Scale bar, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 3.

TSPO deletion in Leydig and Sertoli cells does not affect spermatogenesis. A, Immunohistochemical (IHC) localization showing complete absence of TSPO in Leydig and Sertoli cells of TSPOcΔ/Δ testes. Hematoxylin and eosin (H&E) staining showing unaltered seminiferous tubule morphology and spermatogenesis in TSPOcΔ/Δ testes (n = 5). Scale bars, 50 μm. B, Western blot showing absence of TSPO in TSPOcΔ/Δ testis tissue (n = 5); β-actin is shown as the loading control. C, Cauda epididymal sperm counts were not significantly different between TSPOfl/fl and TSPOcΔ/Δ mice (mean ± SEM; n = 5/group). D–F, Testis cDNA from TSPOfl/fl and TSPOcΔ/Δ mice examined for amplification products from exons 1 and 2 [250 bp] (D); exons 2 and 3 [241 bp] (E); exons 3 and 4 [424 bp] (F); exons 1–4 [711 bp in TSPOfl/fl and 361 bp in TSPOcΔ/Δ]. For all RT-PCR, glyceraldehydes-3-phosphate dehydrogenase was used as a control (CON).

Figure 4.

TSPO deletion in Leydig and Sertoli cells does not affect testosterone production. A, Plasma testosterone levels were not significantly different between TSPOfl/fl and TSPOcΔ/Δ mice (n = 19–22/group). B, When sampled 1 hour after hCG stimulation, plasma testosterone levels were highly elevated but not different between TSPOfl/fl and TSPOcΔ/Δ mice (n = 7/group). C, A modest but significant increase in testis weights was observed in TSPOcΔ/Δ mice compared with TSPOfl/fl mice (P < .05; mean ± SEM; n = 18/group). D, Seminal vesicle weights were not significantly different between TSPOfl/fl and TSPOcΔ/Δ mice (mean ± SEM; n = 18/group).

Figure 5.

StAR expression is unchanged in TSPOcΔ/Δ testis. A, Representative Western blot showing no change in testicular StAR protein in TSPOcΔ/Δ compared with TSPOfl/fl mice; β-actin is shown as the loading control. B, Relative intensity of testicular StAR protein expression (ratios of StAR/β-actin band intensities) between TSPOfl/fl and TSPOcΔ/Δ mice was not significantly different (n = 3/group).

Figure 6.

TSPO deletion does not affect expression of genes involved in testicular steroidogenesis. A, TSPO expression was undetectable in TSPOcΔ/Δ testis. StAR (B), CYP11A1 (C), and HSDB1 (D) expression levels were similar between TSPOfl/fl and TSPOcΔ/Δ testes. E, TSPO2 expression was not detectable in TSPOfl/fl and TSPOcΔ/Δ testes. Femur bone marrow was used as a positive control. (mean ± SEM; ND*, not detected; n = 6/group)