Translocator protein 18 kDa (TSPO; previously known as peripheral benzodiazepine receptor PBR) is a high affinity drug- and cholesterol-binding protein strongly expressed in steroidogenic cells, including testicular Leydig cells, where it is located at the outer mitochondrial membrane (OMM) [1
]. TSPO plays an essential role in cholesterol transfer from the outer to the inner mitochondrial membrane, where CYP11A1 catalyzes pregnenolone formation from cholesterol, the first step of the steroidogenic cascade [1
]. Although present mainly as monomers, TSPO can form polymers upon hormonal stimulation and oxidative stress, and is also found as polymers in tumorigenic cells [2
]. In addition to steroidogenic tissues, TSPO is expressed in multiple tissues and cells, where it is involved in various processes, including cellular proliferation, apoptosis, transport, and differentiation [4
]. While the role of TSPO in steroidogenic cells has been well documented, its role in non-steroidogenic cells is not well understood.
We previously made the serendipitous observation that TSPO was present in neonatal rat gonocytes (also known as pre- and pro-spermatogonia), the precursor cells of type A spermatogonia, including cells of the first spermatogenic wave and spermatogonial stem cells (SSCs) [8
]. In gonocytes, TSPO expression was nuclear, and its drug ligand did not regulate gonocyte proliferation [8
]. TSPO was also found in pachytene spermatocytes and dividing spermatogonia in adult rat testis [8
]. In a recent study, we found that TSPO transcripts are also abundant in adult mouse spermatogonia, pachytene spermatocytes and round spermatids, while the protein is observed in mouse sperm [9
Adequate gonocyte development is critical for the establishment of a SSC reservoir that will support the production of sperm throughout adulthood [10
]. These transitional cells undergo proliferation and migration to the basement membrane of the seminiferous cords, prior to their differentiation [10
]. We have previously shown that gonocyte proliferation is stimulated by the coordinated action of platelet-derived growth factor (PDGF)-BB and 17β-estradiol, and requires ERK1/2 pathway activation [13
]. We also found that gonocyte differentiation is stimulated by all-trans retinoic acid (RA) and involves the activation of PDGFR, SRC and JAK2/STAT5 pathways, highlighting the complexity of this process [15
]. Another important regulatory process is the apoptosis of gonocytes that failed to migrate and/or differentiate, taking place at the end of the first postnatal week [17
]. Studies have suggested that improper gonocyte development may lead to infertility and testicular germ cell tumors (TGCTs), a reproductive pathology on the rise in the past decades [18
]. Thus, better understanding of gonocyte development should elucidate the mechanisms leading to germline stem cell formation, as well as the origins of TGCTs.
In the present study, we examined whether TSPO is altered during differentiation in gonocytes and in the F9 embryonal carcinoma cell line, a mouse TGCT that has retained embryonic stem cell properties, as well as somatic and germ cell markers [16
]. TSPO’s potential role in RA-induced gonocyte differentiation was examined by silencing its expression. TSPO expression profiles were also determined in human adult normal testis and TGCTs. Together, these studies suggest that TSPO is regulated during germ cell differentiation and in germ cell tumors.
TSPO is a ubiquitous and multifunctional protein [4
], shown by various pharmacological and molecular means to play a critical role in steroidogenesis [26
]. While TSPO main location in testis is in the mitochondria of steroidogenic Leydig cells, we have found in previous studies that TSPO is also expressed in rat and mouse male germ cells in an age and spermatogenic cycle dependent manner [8
]. The goal of the present study was to examine if TSPO plays a role in gonocyte differentiation, an essential process at the basis of spermatogenesis, and to examine its patterns of expression in healthy human testicular germ cells and in seminomas, which are thought to arise from disrupted gonocyte differentiation [18
Our initial finding that gonocytes express higher levels of TSPO than PND8 spermatogonia led us to speculate that TSPO expression is down-regulated during gonocyte differentiation. Moreover, the change in TSPO subcellular localization from nuclear in gonocytes to cytoplasmic in spermatogonia is reminiscent of other factors translocating in spermatogenic cells from one compartment to another in function of their activation status. For example, FOXO1 has been shown to translocate from cytoplasm to nucleus between PND1 and 3, a period corresponding to the transition from quiescence to mitosis, and to remain nuclear in SSC [28
]. TSPO nuclear localization has been reported before, particularly in breast cancer cells, where it is involved in cell proliferation [4
]. However, this does not seem to be the case in gonocytes, since adding a TSPO binding drug to gonocytes in presence or absence of proliferation factors did not have an effect [8
To examine whether TSPO is down-regulated during gonocyte differentiation, isolated PND3 gonocytes were treated with the differentiation factor RA. Our data confirmed that RA exerts a repressive effect on the expression of TSPO mRNA and protein during gonocyte differentiation, a process characterized by an increase in Stra8 expression. Interestingly, TSPO promoter has been shown to contain putative binding sites for RARβ and RARγ [7
]. Together with our previous data showing that PND3 gonocytes express high levels of RARγ [16
], this suggests that RARγ activation may be involved in TSPO down-regulation.
Similar effects were observed in F9 mouse embryonal carcinoma cells, a cell line issued from a mouse testicular teratoma, which shares a number of pluripotency markers with embryonic stem cells as well as gonocytes [15
]. In F9 cells, which express genes from both the somatic and germline lineages, RA treatment can induce the expression of markers of somatic (e.g., collagen IV) and germ cell (e.g., Stra8) fates, depending of the culture conditions and downstream pathway activated [16
]. All TGCTs, including embryonal carcinoma, are believed to derive from a common precursor, the carcinoma in situ, itself resulting from the failed differentiation of gonocytes [18
]. Therefore, one cannot exclude at present that RA-induced TSPO repression in F9 cells may correspond to a function retained from gonocytes, which was not altered during the process of carcinogenesis.
To better understand TSPO role in gonocyte differentiation, we knocked down Tspo
in gonocytes treated or not with RA. Surprisingly, silencing TSPO expression with siRNA was sufficient to significantly increase Stra8
expression in gonocytes, in basal conditions with low levels of RA provided by FBS. Moreover, TSPO knock down potentiated RA-mediated increases in Stra8
transcripts, implying that TSPO represses Stra8 expression. Among its multiple roles, TSPO has been reported to be positively regulated during adipocyte differentiation and homeostasis, with its knockdown impairing the release of adipokines, glucose uptake, and adipogenesis [31
]. We have previously shown that gonocyte differentiation involves the activation of platelet-derived growth factor receptors and downstream SRC, JAK2, and STAT5 pathways [16
] and an active ubiquitin proteasome system [20
]. In contrast to these positive regulators of gonocyte differentiation, the present results suggest that TSPO acts as a repressor on gonocyte differentiation, and that one of RA effects is to eliminate this block. This is reminiscent of the downregulation by RA of Nanos2 in fetal and neonatal gonocytes, where this RNA binding protein was shown to repress Stra8 expression and prevent premature entry of germ cells into meiosis [33
]. It would be interesting to examine in future studies whether TSPO ligands with agonist properties might exert a repressing effect on gonocyte differentiation, as suggested by our silencing results.
We then analyzed the mRNA expression of two germ cell markers, the PIWI-interacting RNA binding proteins Mili, and Miwi2, both required for spermatogenesis and meiotic progression. MILI is expressed from gonocytes to early spermatocytes, and has been shown to be essential for spermatogonial stem cell self-renewal and differentiation [21
]. MIWI2 is strongly expressed in gonocytes, where it is guiding the re-methylation of transposons and preventing their activation, following the global erasure of DNA methylation marks in primordial germ cells [22
]. Besides their role in transposon methylation, these genes have also been implicated in histone modifications and post-transcriptional regulation [34
]. While the expression patterns of the two genes did not appear to be regulated by RA, TSPO knockdown led to increases in both Mili
transcripts, suggesting TSPO involvement in the down-regulation of these genes, independent of differentiation signals. While preserving the genomic integrity of germ cells is critical for species survival, allowing for some degree of transposon mobility has played a role in evolution and adaptability of species to environmental changes. Considering the multiple roles of TSPO, the fact that it is highly conserved throughout evolution from bacteria to human [35
], and its nuclear localization in gonocytes, one could envision a role for TSPO in maintaining the balance between transposon mobility and preserving genomic integrity in gonocytes. Further experiments will be required to test this hypothesis.
Next, we examined whether TSPO is also expressed in human normal and pathological germ cells, which had not been reported in the literature. Our study showed that normal testicular tissues express TSPO in discreet phases of spermatogenic cells, similarly to our previous findings in rat and mouse testis [8
]. In human testis, besides its expected high expression in Leydig cells, TSPO was clearly expressed in the forming acrosomes of round spermatids, as confirmed by co-localization with PNA, a peanut agglutinin lectin, commonly used as an acrosomal marker [23
]. This is a novel finding, suggesting that TSPO is involved in the formation of specialized organelles in germ cells. Interestingly, TSPO has been previously reported to be expressed in the Golgi apparatus of liver cells, suggesting that it might play a similar role in somatic cells [36
Finally, in our search of TSPO role in germ cells, we examined its expression patterns in seminoma, the most common testicular germ cell tumor type in human, proposed to originate from abnormal gonocytes [18
]. The comparative study of several normal and seminoma samples revealed a significant upregulation in TSPO
mRNA levels in seminomas. Immunofluorescent analysis of TSPO and two pluripotency transcription factors OCT3/4 and AP2γ used as seminoma markers [24
] confirmed TSPO protein localization in seminoma cells. Several studies have reported changes in TSPO expression and potential role in various cancers. For example, TSPO expression is upregulated in glioma tumor cells, compared to normal brain [37
]. TSPO has also been implicated in lung cancer development, as cigarette smoke exposure was found to alter TSPO protein, leading the way for the initiation and progression of lung cancer [38
]. Studies have shown that TSPO is highly expressed in estrogen-receptor (ER) negative breast tumors, representing a potential target for the development of new therapies for this subset of breast cancer [39
]. Moreover, studies have shown that that TSPO expression correlates positively with the invasiveness and/or malignancy of breast, colorectal and prostate cancers [4
]. Thus, our study revealed one more type of cancer presenting TSPO dysregulation. Determining whether TSPO alterations reflect its involvement in testicular tumorigenesis process, or if they indicate the retention of an early germ cell marker in deficient germ cells remains to be determined.
Taken together, our study shows that TSPO is down regulated during gonocyte differentiation, in which it might play a repressive role. Moreover, our expression studies in human normal testes confirmed that TSPO is expressed in subsets of adult germ cells, suggesting a function in acrosome formation, while the analysis of tumor samples revealed its mRNA up-regulation and protein localization in seminoma cells. Further studies are needed to determine the exact role of TSPO in normal spermatogenic cell development, from gonocyte to more mature germ cells, and in testicular cancer.