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...

Gonad-stimulating substance (GSS) of starfish is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to the vertebrate luteinizing hormone (LH). Here, we purified GSS of starfish, Asterina pectinifera, from radial nerves and determined its amino acid sequence. The purified GSS was a heterodimer composed of 2 different peptides, A and B chains, with disulfide cross-linkages. Based on its cysteine motif, starfish GSS was classified as a member of the insulin/insulin-like growth factor (IGF)/relaxin superfamily. The cDNA of GSS encodes a preprohormone sequence with a C peptide between the A and B chains. Phylogenetic analyses revealed that starfish GSS was a relaxin-like peptide. Chemically synthesized GSS induced not only oocyte maturation and ovulation in isolated ovarian fragments, but also unique spawning behavior, followed by release of gametes shortly after the injection. Importantly, the action of the synthetic GSS on oocyte maturation and ovulation was mediated through the production of cAMP by isolated ovarian follicle cells, thereby producing the maturationinducing hormone of this species, 1-methyladenine. In situ hybridization showed the transcription of GSS to occur in the periphery of radial nerves at the side of tube feet. Together, the structure, sequence, and mode of signal transduction strongly suggest that GSS is closely related to the vertebrate relaxin.gonadotropin ͉ gonad-stimulating substance ͉ insulin-like growth factor/relaxin superfamily ͉ 1-methyladenine ͉ in situ hybridization

Round spermatids were prepared from rat testes and incubated with various substrates (glucose, fructose, pyruvate, lactate and acetate) to measure utilization of substrates and production of ATP in the presence of saturating levels of each substrate. By both criteria lactate is the preferred substrate by a factor of 3 or 4. Production of more than half of the ATP with lactate is substrate is prevented by addition of an inhibitor of alpha-ketoacid dehydrogenase (5-methoxyindole-2-carboxylic acid) Pyruvate and lactate are interconverted and pyruvate inhibits production of ATP from lactate. Synthesis of ATP with lactate and with pyruvate is inhibited by rotenone, rutamycin or 2,4-dinitrophenol. Utilization of glucose is limited by aldolase activity. These findings suggest that exogenous lactate is oxidized by lactate dehydrogenase followed by pyruvate dehydrogenase and Krebs; cycle enzymes under conditions which do not allow pyruvate to inhibit lactate dehydrogenase. ATP is synthesized through electron transport. Post-mitochondrial supernate from spermatids showed that high concentration of pyruvate (greater than 1 mM) inhibit lactate dehydrogenase with pyruvate as substrate and that with lactate as substrate, pyruvate behaves as a competitive inhibitor of lactate dehydrogenase. Evidently lactate is the preferred substrate for round spermatids and energy production is most efficient when this substance is present in high concentrations and pyruvate is present in low concentrations. Reasons are given for suggesting that Sertoli cells may provide the relatively large amounts of lactate required by round spermatids.

Cultured Sertoli cells prepared from young rats (13 days old) showed increased incorporation of [3H]thymidine into DNA, increased production of lactate, and increased incorporation of [3H]leucine into protein in response to micromolar concentrations of insulin and nanomolar concentrations of insulin-like growth factor II (IGF-II). The first of these responses was also seen with nanomolar concentrations of IGF-I. Receptor affinity labeling studies using Sertoli cell membranes and whole Sertoli cells showed that these cells possess abundant growth factor receptors of type I (mol wt, 350,000) that show high affinity for IGF-I, moderate affinity for IGF-II, and low affinity for insulin. Sertoli cell membranes also show abundant growth factor receptors of type II (mol wt, 230,000) that show high affinity for IGF-II, moderate affinity for IGF-I, and no detectable affinity for insulin. Moreover, the responses of the Sertoli cell to insulin were observed at concentrations of 100 nM or higher, whereas insulin receptors are known to be saturated by insulin at concentrations of 10 nM or less. It is, therefore, concluded that Sertoli cells possess receptors for IGF-I and that the responses observed to insulin may result from binding of these hormones to receptors for IGF-I.

Gamete maturation and spawning in starfish is triggered by a gonad‐stimulating substance (GSS), which is present in extracts of the radial nerve cords. Purification of GSS from the starfish Patiria pectinifera identified GSS as a relaxin‐like polypeptide, which is now known as relaxin‐like gonad‐stimulating peptide (RGP). Cells expressing RGP in the radial nerve cord of P. pectinifera have been visualized, but the presence of RGP‐expressing cells in other parts of the starfish body has not been investigated. Here we addressed this issue in the starfish Asterias rubens. An A. rubens RGP (AruRGP) precursor cDNA was sequenced and the A chain and B chain that form AruRGP were detected in A. rubens radial nerve cord extracts using mass spectrometry. Comparison of the bioactivity of AruRGP and P. pectinifera RGP (PpeRGP) revealed that both polypeptides induce oocyte maturation and ovulation in A. rubens ovarian fragments, but AruRGP is more potent than PpeRGP. Analysis of the expression of AruRGP in A. rubens using mRNA in situ hybridization revealed cells expressing RGP in the radial nerve cords, circumoral nerve ring, and tube feet. Furthermore, a band of RGP‐expressing cells was identified in the body wall epithelium lining the cavity that surrounds the sensory terminal tentacle and optic cushion at the tips of the arms. Discovery of these RGP‐expressing cells closely associated with sensory organs in the arm tips is an important finding because these cells are candidate physiological mediators for hormonal control of starfish spawning in response to environmental cues. J. Comp. Neurol. 525:1599–1617, 2017. © 2016 Wiley Periodicals, Inc.