Conditional steroidogenic cell-targeted deletion of TSPO unveils a crucial role in viability and hormone-dependent steroid formation

Abstract

Translocator protein (TSPO) is a key member of the mitochondrial cholesterol transport complex in steroidogenic tissues. To assess the function of TSPO, we generated two lines of Cre-mediated Tspo conditional knockout (cKO) mice. First, gonadal somatic cell-targeting Amhr2-Cre mice were crossed with Tspo-floxed mice to obtain F1 Tspo Amhr2 cKO mice (Tspo(fl/fl);Amhr2-Cre(/+)). The unexpected Mendelian ratio of 4.4% cKO mice was confirmed by genotyping of 12.5-day-postcoitum (dpc) embryos. As Amhr2-Cre is expressed in gonads at 12.5 dpc, these findings suggest preimplantation selection of embryos. Analysis of expression databases revealed elevated levels of Amhr2 in two- and eight-cell zygotes, suggesting ectopic Tspo silencing before the morula stage and demonstrating elevated embryonic lethality and involvement of TSPO in embryonic development. To circumvent this issue, steroidogenic cell-targeting Nr5a1-Cre mice were crossed with Tspo-floxed mice. The resulting Tspo(fl/fl);Nr5a1-Cre(/+) mice were born at a normal Mendelian ratio. Nr5a1-driven Tspo cKO mice exhibited highly reduced Tspo levels in adrenal cortex and gonads. Treatment of mice with human chorionic gonadotropin (hCG) resulted in increased circulating testosterone levels despite extensive lipid droplet depletion. In contrast, Nr5a1-driven Tspo cKO mice lost their ability to form corticosterone in response to adrenocorticotropic hormone (ACTH). Important for ACTH-dependent steroidogenesis, Mc2r, Stard1, and Cypa11a1 levels were unaffected, whereas Scarb1 levels were increased and accumulation of lipid droplets was observed, indicative of a blockade of cholesterol utilization for steroidogenesis. TSPO expression in the adrenal medulla and increased epinephrine production were also observed. In conclusion, TSPO was found necessary for preimplantation embryo development and ACTH-stimulated steroid biosynthesis.

Keywords: anti-Mullerian hormone receptor type II; knockout mice; nuclear receptor subfamily 5 group A member 1; steroidogenesis; translocator protein.

Fig. 1.

Breeding scheme and abnormal Mendelian ratio observed in Tspo-deficient mice (Cre^+/−; Tspo^fl/fl). (A) Breeding scheme: WT, Cre-positive, and Cre-negative Tspo homofloxed mice were generated and mated to generate Tspo tissue-specific HE mice, expected to represent 25% of the offspring. The second round of breeding that crossed HE mice with WT Tspo homofloxed mice was expected to produce 25% Tspo tissue-specific HO mice. (B) Mendelian ratio of Amhr2-Cre × Tspo homofloxed mice from 180 mice. The observed values (blue bars) are compared with the theoretical values (red bars) for male and female. χ²-Test; ***P < 0.001; n.s., not significant; n = 180. (C) Morphology of an embryo at 12.5 dpc and one uterus extirpated on 12.5 dpc. (D) Genotyping of 22 representative embryos from two uteri. (Top) PCR showing which embryos are Cre-positive. (Bottom) PCR showing WT, HE, or HO Tspo floxed genotypes. Rare cases of Cre-positive Tspo homofloxed embryo (red arrow) and Cre-positive Tspo WT embryo (blue arrow) are indicated. (E) Bar graph of the Mendelian and sex ratios of 48 embryos showing the expected ratios (red bars) and experimental results (blue bars). χ²-Test; ***P < 0.001; n = 48. (F) Expression profile of the Amhr2 gene during preimplantation mouse embryo development. The scatter plot of the Affymetrix reading of Amhr2 gene using probe 1457021_×_at is shown against each preimplantation developmental stage: oocyte, 1 cell, 2 cells, 8 cells, and blastocyst. Affymetrix data were retrieved from GSE1749 with the platform Affymetrix mouse expression 430B array. n = 4.

Fig. 2.

Conditional reduction of Tspo mRNA levels and TSPO protein expression in WT, HE, and HO mice. (A and B) Real-time PCR of Tspo mRNA expression and confocal immunofluorescence images of TSPO protein in mouse testis (A and B), male adrenal gland (C and D), and ovary (E and F). Arrows point to Leydig cell (Lc). C, cortex; M, medulla. T, theca cells; G, granulosa cells. The β-actin (Actb) gene was used for normalization of real-time PCR data. In the confocal microscopy images, red indicates anti-TSPO immunoreactivity, and blue indicates DAPI staining. [Scale bar: 100 μm (A, B, E, and F); 50 μm (C and D).] Student’s t test; **P < 0.01; ***P < 0.001.

Fig. 3.

Circulating testosterone and corticosterone levels in WT, HE, and HO Tspo cKO mice treated with and without hCG or ACTH. (A) Plasma testosterone levels from basal and hCG-treated WT, HE, and HO mice. No significant differences compared with the control were found. Mann–Whitney U test, P > 0.05; n = 14–20 animals per group. (B) Plasma corticosterone levels from basal and ACTH-treated WT, HE, and HO mice. Mann–Whitney U test; *P < 0.05, **P < 0.01; n = 10–13 animals per group. Lower plots show basal and ACTH-induced corticosterone levels in individual WT, HE, and HO mice.

Fig. 4.

Effect of Tspo deficiency on mRNA levels of steroidogenic genes and esterified cholesterol. (A and B) Real-time PCR of mRNA expression of Star, Cyp11a1, Mc2r, and Scarb1 in testes (A) and adrenal glands (B). The β-actin mRNA (Actb) was used for normalization. Student’s t test; *P < 0.01. (C) Oil Red O staining of testes from WT, HE, and HO mice. HO mice exhibited a notable decrease in Oil Red O staining of neutral lipids. Lc, Leydig cells. (Scale bar: 200 μm.) (D and E) Oil Red O staining of adrenal glands in WT, HE, and HO mice. A notable increase in accumulation of neutral lipid staining is observed as highlighted in the masked image, created using Image-Pro Plus software on the images shown in D. C, cortex. (Scale bar: 200 μm.)