Kanako Morohaku

Translocator Protein/Peripheral Benzodiazepine Receptor Is Not Required for Steroid Hormone Biosynthesis

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.

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.

Complete in vitro generation of fertile oocytes from mouse primordial germ cells

Significance Throughout the life of female mammals, only a small number of viable oocytes are produced. The mechanisms underlying the creation and selection of competent oocytes remain unclear. Here, we propose a novel approach for elucidating these unsolved questions, involving the use of an in vitro system established in the present study, which can fully reproduce mammalian oogenesis from mouse fetal primordial germ cells. Reconstitution of the entire oogenesis process has not been previously accomplished. Our system will assist in understanding the mechanisms of oogenesis and also create a new gamete resource in mammals. Reconstituting gametogenesis in vitro is a key goal for reproductive biology and regenerative medicine. Successful in vitro reconstitution of primordial germ cells and spermatogenesis has recently had a significant effect in the field. However, recapitulation of oogenesis in vitro remains unachieved. Here we demonstrate the first reconstitution, to our knowledge, of the entire process of mammalian oogenesis in vitro from primordial germ cells, using an estrogen-receptor antagonist that promotes normal follicle formation, which in turn is crucial for supporting oocyte growth. The fundamental events in oogenesis (i.e., meiosis, oocyte growth, and genomic imprinting) were reproduced in the culture system. The most rigorous evidence of the recapitulation of oogenesis was the birth of fertile offspring, with a maximum of seven pups obtained from a cultured gonad. Moreover, cryopreserved gonads yielded functional oocytes and offspring in this culture system. Thus, our in vitro system will enable both innovative approaches for a deeper understanding of oogenesis and a new avenue to create and preserve female germ cells.

A brief history of the search for the protein(s) involved in the acute regulation of steroidogenesis.

The synthesis of steroid hormones occurs in specific cells and tissues in the body in response to trophic hormones and other signals. In order to synthesize steroids de novo, cholesterol, the precursor of all steroid hormones, must be mobilized from cellular stores to the inner mitochondrial membrane (IMM) to be converted into the first steroid formed, pregnenolone. This delivery of cholesterol to the IMM is the rate-limiting step in this process, and has long been known to require the rapid synthesis of a new protein(s) in response to stimulation. Although several possibilities for this protein have arisen over the past few decades, most of the recent attention to fill this role has centered on the candidacies of the proteins the Translocator Protein (TSPO) and the Steroidogenic Acute Regulatory Protein (StAR). In this review, the process of regulating steroidogenesis is briefly described, the characteristics of the candidate proteins and the data supporting their candidacies summarized, and some recent findings that propose a serious challenge for the role of TSPO in this process are discussed.

Mitochondrial translocator protein (TSPO) function is not essential for heme biosynthesis

Function of the mammalian translocator protein (TSPO; previously known as the peripheral benzodiazepine receptor) remains unclear because its presumed role in steroidogenesis and mitochondrial permeability transition established using pharmacological methods has been refuted in recent genetic studies. Protoporphyrin IX (PPIX) is considered a conserved endogenous ligand for TSPO. In bacteria, TSPO was identified to regulate tetrapyrrole metabolism and chemical catalysis of PPIX in the presence of light, and in vertebrates, TSPO function has been linked to porphyrin transport and heme biosynthesis. Positive correlation between high TSPO expression in cancer cells and susceptibility to photodynamic therapy based on their increased ability to convert the precursor 5-aminolevulinic acid (ALA) to PPIX appeared to reinforce this mechanism. In this study, we used TSPO knock-out (Tspo−/−) mice, primary cells, and different tumor cell lines to examine the role of TSPO in erythropoiesis, heme levels, PPIX biosynthesis, phototoxic cell death, and mitochondrial bioenergetic homeostasis. In contrast to expectations, our results demonstrate that TSPO deficiency does not adversely affect erythropoiesis, heme biosynthesis, bioconversion of ALA to PPIX, and porphyrin-mediated phototoxic cell death. TSPO expression levels in cancer cells do not correlate with their ability to convert ALA to PPIX. In fibroblasts, we observed that TSPO deficiency decreased the oxygen consumption rate and mitochondrial membrane potential (ΔΨm) indicative of a cellular metabolic shift, without a negative impact on porphyrin biosynthetic capability. Based on these findings, we conclude that mammalian TSPO does not have a critical physiological function related to PPIX and heme biosynthesis.

Developmental Expression of Translocator Protein/Peripheral Benzodiazepine Receptor in Reproductive Tissues

Translocator protein (TSPO) present in the outer mitochondrial membrane has been suggested to be critical for cholesterol import, a rate-limiting step for steroid hormone biosynthesis. Despite the importance of steroidogenesis in regulating reproductive functions, the developmental profile of TSPO expression in the gonads and accessory sex organs has not been completely characterized. As a first step towards understanding the function of TSPO, we studied its expression in male and female murine reproductive organs. We examined testes and ovaries at embryonic days 14.5 and 18.5, and postnatal days 0, 7, 14, 21 and 56 of development. In the adult testis, TSPO was expressed in both Leydig cells and Sertoli cells. In the developing testes TSPO expression was seen in immature Sertoli cells, fetal Leydig cells and gonocytes. In the ovary, TSPO was expressed in the ovarian surface epithelium, interstitial cells granulosa cells and luteal cells. Corpora lutea of ovaries from pregnant mice showed strong expression of TSPO. In the developing ovary, TSPO expression was seen in the squamous pregranulosa cells associated with germ line cysts, together with progressively increasing expression in interstitial cells and the ovarian surface epithelium. In adult mice, the epithelia of other reproductive tissues like the epididymis, prostate, seminal vesicle, oviduct and uterus also showed distinct patterns of TSPO expression. In summary, TSPO expression in both male and female reproductive tissues was not only restricted to steroidogenic cells. Expression in Sertoli cells, ovarian surface epithelium, efferent ductal epithelium, prostatic epithelium, seminal vesiclular epithelium, uterine epithelium and oviductal epithelium suggest either previously unknown sites for de novo steroidogenesis or functions for TSPO distinct from its well-studied role in steroid hormone production.

Incorporation of phosphatase inhibitor in culture prompts growth initiation of isolated non-growing oocytes.

In vitro folliculogenesis of primordial and early preantral follicles is necessary for increment of reproductive efficiency in domestic animals, humans and endangered species. Recent study in phosphatase and tensin homolog (Pten) -knockout mice has revealed that this phosphatase acts as an inhibitory factor in follicle activation of primordial pool with the resultant inhibition of oocyte growth. To test in vitro effect of a phosphatase inhibitor on growth initiation of isolated non-growing oocytes in neonatal ovaries, we applied a specific inhibitor (bpV (HOpic)) for PTEN in culturing system. Non-growing oocytes isolated from the ovaries of newborn BDF1 (C57BL/6 × DBA/2) pups were divided to four culture groups. Five days after culture, the oocytes in 14 μmol/l bpV only, 14 μmol/l bpV plus 100 ng/ml Kit Ligand (KL), and 100 ng/ml KL groups showed significantly (P<0.05) growth (19.3±0.55, 25.8±0.53 and 21.6±0.29 μm, respectively) compared with that of the control (no additive) (16.9±0.53 μm). In addition, western blotting in those groups showed enhanced expression of phosphorylated Akt. In conclusion, we clearly demonstrate that isolated non-growing oocytes develop in phosphatase inhibitor, especially to PTEN, incorporated culturing system, and show first as we know that oocytes with zona Pellucidae can be obtained in vitro from isolated non-growing oocytes.

Developmental competence of oocytes grown in vitro: Has it peaked already?

In vitro growth of immature oocytes provides opportunities to increase gametic resources and to understand the mechanisms underlying oocyte development. Many studies on the in vitro growth of oocytes have been reported thus far; however, only a few cases have been reported, which demonstrated that oocytes can support full-term development after in vitro fertilization. Our research group recently found that culture of mouse neonatal primordial follicles increased the birthrate; however, the establishment of an in vitro system that can completely mimic follicle or oocyte growth in vivo and control oogenesis remains an ongoing challenge.

Development of fertile mouse oocytes from mitotic germ cells in vitro

Mammalian fetal ovaries contain numerous primordial germ cells (PGCs), although few mature oocytes are obtained from females, owing to apoptosis and follicle atresia. The regulatory mechanisms underlying oogenesis/folliculogenesis remain unknown. Development of methods for obtaining mature oocytes from PGCs in fetal ovaries in vitro could contribute to clarifying these mechanisms. The failure of follicle assembly has been found to be the most challenging aspect in conventional culture conditions. Recently, we established novel culture conditions that enable successful follicle assembly, sustaining interactions between the oocyte and somatic cells, and, in turn, promoting oocyte growth and maturation. Mature oocytes were differentiated from PGCs after a 1-month culture period. A hundred mouse offspring were obtained from approximately a thousand mature oocytes, indicating that oocytes that were differentiated from PGCs in vitro acquired totipotency after fertilization. Here we provide a detailed protocol for using this in vitro system. This in vitro system will potentially provide a novel platform for studying oogenesis and preservation of female germ cells.