Malfunction of the circadian clock has been linked to the pathogenesis of a variety of diseases. We show that mice lacking the core clock components Cryptochrome-1 (Cry1) and Cryptochrome-2 (Cry2) (Cry-null mice) show salt-sensitive hypertension due to abnormally high synthesis of the mineralocorticoid aldosterone by the adrenal gland. An extensive search for the underlying cause led us to identify type VI 3beta-hydroxyl-steroid dehydrogenase (Hsd3b6) as a new hypertension risk factor in mice. Hsd3b6 is expressed exclusively in aldosterone-producing cells and is under transcriptional control of the circadian clock. In Cry-null mice, Hsd3b6 messenger RNA and protein levels are constitutively high, leading to a marked increase in 3beta-hydroxysteroid dehydrogenase-isomerase (3beta-HSD) enzymatic activity and, as a consequence, enhanced aldosterone production. These data place Hsd3b6 in a pivotal position through which circadian clock malfunction is coupled to the development of hypertension. Translation of these findings to humans will require clinical examination of human HSD3B1 gene, which we found to be functionally similar to mouse Hsd3b6.

Melatonin regulates diurnal changes in locomotor activity in vertebrates, but the molecular mechanism for this neurohormonal regulation of behavior is poorly understood. Here we show that 7␣-hydroxypregnenolone, a previously undescribed avian neurosteroid, mediates melatonin action on diurnal locomotor rhythms in quail. In this study, we first identified 7␣-hydroxypregnenolone and its stereoisomer 7␤-hydroxypregnenolone in quail brain. These neurosteroids have not been described previously in avian brain. We then demonstrated that 7␣-hydroxypregnenolone acutely increased quail locomotor activity. To analyze the production of 7␣-hydroxypregnenolone, cytochrome P450 7␣ , a steroidogenic enzyme of this neurosteroid, was also identified. Subsequently, we demonstrated diurnal changes in 7␣-hydroxypregnenolone synthesis in quail. 7␣-Hydroxypregnenolone synthesis and locomotor activity in males were much higher than in females. This is the first demonstration in any vertebrate of a clear sex difference in neurosteroid synthesis. This sex difference in 7␣-hydroxypregnenolone synthesis corresponded to the sex difference in locomotion. We show that only males exhibited marked diurnal changes in 7␣-hydroxypregnenolone synthesis, and these changes occurred in parallel with changes in locomotor activity. Finally, we identified melatonin as a key component of the mechanism regulating 7␣-hydroxypregnenolone synthesis. Increased synthesis of 7␣-hydroxypregnenolone occurred in males in vivo after melatonin removal via pinealectomy and orbital enucleation (Px plus Ex). Conversely, decreased synthesis of this neurosteroid occurred after melatonin administration to Px plus Ex males. This study demonstrates that melatonin regulates synthesis of 7␣-hydroxypregnenolone, a key factor for induction of locomotor activity, thus inducing diurnal locomotor changes in male birds. This is a previously undescribed role for melatonin.

It is believed that neurosteroids are produced in the brain and other nervous systems. Here, we show that allopregnanolone (ALLO), a neurosteroid, is exceedingly produced in the pineal gland compared with the brain and that pineal ALLO acts on the Purkinje cell, a principal cerebellar neuron, to prevent apoptosis in the juvenile quail. We first demonstrated that the pineal gland is a major organ of neurosteroidogenesis. A series of experiments using molecular and biochemical techniques has further demonstrated that the pineal gland produces a variety of neurosteroids de novo from cholesterol in the juvenile quail. Importantly, ALLO was far more actively produced in the pineal gland than in the brain. Pinealectomy (Px) decreased ALLO concentration in the cerebellum and induced apoptosis of Purkinje cells, whereas administration of ALLO to Px quail chicks prevented apoptosis of Purkinje cells. We further found that Px significantly increased the number of Purkinje cells that expressed active caspase-3, a key protease in apoptotic pathway, and daily injection of ALLO to Px quail chicks decreased the number of Purkinje cells expressing active caspase-3. These results indicate that the neuroprotective effect of pineal ALLO is associated with the decrease in caspase-3 activity during the early stage of neuronal development. We thus provide evidence that the pineal gland is an important neurosteroidogenic organ and that pineal ALLO may be involved in Purkinje cell survival during development. This is an important function of the pineal gland in the formation of neuronal circuits in the developing cerebellum.steroid | melatonin | HPLC | gas chromatography/mass spectrometry T he cerebellar cortex has been used as an excellent model to study synaptic formation and transmission of neural networks because it forms relatively simple neuronal networks compared with those of other brain regions. The Purkinje cell is a principal cerebellar neuron that integrates the process of memory and learning. It is known that in birds and mammals pinealectomy (Px) induces cell loss in the brain including Purkinje cells during development (1, 2). This observation suggests that a certain component(s) in the pineal gland contributes to Purkinje cell survival during development.It is now established that steroids can be synthesized de novo in the central and peripheral nervous systems. Such steroids are called "neurosteroids," and de novo neurosteroidogenesis from cholesterol is a conserved property of the vertebrate brain (for reviews, see refs. 3-7). The Purkinje cell is known as a site of neurosteroidogenesis in the brain (for review, see ref. 8). This cerebellar neuron produces progesterone (PROG) and estradiol-17β (E2) de novo from cholesterol during neonatal life, when cerebellar neuronal circuit formation occurs. Both PROG and E2 promote dendritic growth, spinogenesis, and synaptogenesis via each cognate nuclear receptor in the developing Purkinje cell (9-11). Allopregnanolone (ALLO; 3α,5α-tetrahydroprogesterone), a progesterone metabol...

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.