Testosterone is a final product of androgenic hormone biosynthesis, and Leydig cells are known to be the primary source of androgens. In the mammalian testis, two distinct populations of Leydig cells, the fetal and the adult Leydig cells, develop sequentially, and these two cell types differ both morphologically and functionally. It is well known that the adult Leydig cells maintain male reproductive function by producing testosterone. However, it has been controversial whether fetal Leydig cells can produce testosterone, and the synthetic pathway of testosterone in the fetal testis is not fully understood. In the present study, we generated transgenic mice in which enhanced green fluorescence protein was expressed under the control of a fetal Leydig cell-specific enhancer of the Ad4BP/SF-1 (Nr5a1) gene. The transgene construct was prepared by mutating the LIM homeodomain transcription factor (LHX9)-binding sequence in the promoter, which abolished promoter activity in the undifferentiated testicular cells. These transgenic mice were used to collect highly pure fetal Leydig cells. Gene expression and steroidogenic enzyme activities in the fetal Leydig cells as well as in the fetal Sertoli cells and adult Leydig cells were analyzed. Our results revealed that the fetal Leydig cells synthesize only androstenedione because they lack expression of Hsd17b3, and fetal Sertoli cells convert androstenedione to testosterone, whereas adult Leydig cells synthesize testosterone by themselves. The current study demonstrated that both Leydig and Sertoli cells are required for testosterone synthesis in the mouse fetal testis.

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that inhibits gonadotropin secretion and socio-sexual behaviours. Oestrogen (neuroestrogen) synthesized in the brain from androgen by aromatase regulates male socio-sexual behaviours. Here we show that GnIH directly activates aromatase and increases neuroestrogen synthesis in the preoptic area (POA) and inhibits socio-sexual behaviours of male quail. Aromatase activity and neuroestrogen concentration in the POA are low in the morning when the birds are active, but neuroestrogen synthesis gradually increases until the evening when the birds become inactive. Centrally administered GnIH in the morning increases neuroestrogen synthesis in the POA and decreases socio-sexual behaviours. Centrally administered 17β-oestradiol at higher doses also inhibits socio-sexual behaviours in the morning. These results suggest that GnIH inhibits male socio-sexual behaviours by increasing neuroestrogen synthesis beyond its optimum concentration for the expression of socio-sexual behaviours. This is the first demonstration of any hypothalamic neuropeptide that directly regulates neuroestrogen synthesis.

Neurosteroids are synthesizedde novofrom cholesterol in the brain. To understand neurosteroid action in the brain, data on the regio- and temporal-specific synthesis of neurosteroids are needed. Recently, we identified the Purkinje cell as an active neurosteroidogenic cell. In rodents, this neuron actively produces several neurosteroids including estradiol during neonatal life, when cerebellar neuronal circuit formation occurs. Estradiol may be involved in cerebellar neuronal circuit formation through promoting neuronal growth and neuronal synaptic contact, because the Purkinje cell expresses estrogen receptor-β (ERβ). To test this hypothesis, in this study we examined the effects of estradiol on dendritic growth, spinogenesis, and synaptogenesis in the Purkinje cell using neonatal wild-type (WT) mice or cytochrome P450 aromatase knock-out (ArKO) mice. Administration of estradiol to neonatal WT or ArKO mice increased dendritic growth, spinogenesis, and synaptogenesis in the Purkinje cell. In contrast, WT mice treated with tamoxifen, an ER antagonist, or ArKO mice exhibited decreased Purkinje dendritic growth, spinogenesis, and synaptogenesis at the same neonatal period. To elucidate the mode of action of estradiol, we further examined the expression of brain-derived neurotrophic factor (BDNF) in response to estrogen actions in the neonate. Estrogen administration to neonatal WT or ArKO mice increased the BDNF level in the cerebellum, whereas tamoxifen decreased the BDNF level in WT mice similar to ArKO mice. BDNF administration to tamoxifen-treated WT mice increased Purkinje dendritic growth. These results indicate that estradiol induces dendritic growth, spinogenesis, and synaptogenesis in the developing Purkinje cell via BDNF action during neonatal life.

We recently found that the Japanese red-bellied newt, Cynops pyrrhogaster, actively produces 7α-hydroxypregnenolone, a previously undescribed amphibian neurosteroid. 7α-Hydroxypregnenolone stimulates locomotor activity of male newts. Locomotor activity of male newts increases during the breeding period as in other wild animals, but the molecular mechanism for such a change in locomotor activity is poorly understood. Here we show that the adenohypophyseal hormone prolactin (PRL) stimulates 7α-hydroxypregnenolone synthesis in the brain, thus increasing locomotor activity of breeding male newts. In this study, cytochrome P4507α (CYP7B), a steroidogenic enzyme catalyzing the formation of 7α-hydroxypregnenolone, was first identified to analyze seasonal changes in 7α-hydroxypregnenolone synthesis. Only males exhibited marked seasonal changes in 7α-hydroxypregnenolone synthesis and CYP7B expression in the brain, with a maximum level in the spring breeding period when locomotor activity of males increases. Subsequently we identified PRL as a key component of the mechanism regulating 7α-hydroxypregnenolone synthesis. Hypophysectomy decreased 7α-hydroxypregnenolone synthesis in the male brain, whereas administration of PRL but not gonadotropins to hypophysectomized males caused a dose-dependent increase in 7α-hydroxypregnenolone synthesis. To analyze the mode of PRL action, CYP7B and the receptor for PRL were localized in the male brain. PRL receptor was expressed in the neurons expressing CYP7B in the magnocellular preoptic nucleus. Thus, PRL appears to act directly on neurosteroidogenic magnocellular preoptic nucleus neurons to regulate 7α-hydroxypregnenolone synthesis, thus inducing seasonal locomotor changes in male newts. This is the first report describing the regulation of neurosteroidogenesis in the brain by an adenohypophyseal hormone in any vertebrate.