Hormonal control of expression of courtship behavior and of development of structures related to the reproductive behavior in two species of Japanese newts, Cynops pyrrhogaster and Cynops ensicauda, was described. Prolactin (PRL) and androgen were essential factors for eliciting courtship behavior. In addition, arginine vasotocin markedly enhanced the expression of courtship behavior. PRL induced migration to water, in which courtship and oviposition take place, and converted the integument from the terrestrial type to the aquatic one. PRL also stimulated the growth of the tail fin, which was blocked by estrogen. Cellular and nuclear size and number of synapses on the somata of Mauthner cells, which are involved in tail movement, were also increased by PRL and androgen. Synthesis of sodefrin, a female-attracting pheromone, in the abdominal gland as well as that of mucopolysaccharides constituting the sac of sperm in the lateral gland was enhanced by PRL and androgen. Structural development of oviducts was elicited by estrogen or PRL to a certain extent, and full oviducal development by the combination of these two hormones, PRL being indispensable for the oviducal jelly secretion.

Hormonal control of urodele reproductive behavior.

Hormonal control of response to and secretion of sex attractants in Japanese newts.

We recently found that the Japanese red-bellied newt, Cynops pyrrhogaster, actively produces 7alpha-hydroxypregnenolone, a previously undescribed amphibian neurosteroid. 7alpha-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 7alpha-hydroxypregnenolone synthesis in the brain, thus increasing locomotor activity of breeding male newts. In this study, cytochrome P450(7alpha) (CYP7B), a steroidogenic enzyme catalyzing the formation of 7alpha-hydroxypregnenolone, was first identified to analyze seasonal changes in 7alpha-hydroxypregnenolone synthesis. Only males exhibited marked seasonal changes in 7alpha-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 7alpha-hydroxypregnenolone synthesis. Hypophysectomy decreased 7alpha-hydroxypregnenolone synthesis in the male brain, whereas administration of PRL but not gonadotropins to hypophysectomized males caused a dose-dependent increase in 7alpha-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 7alpha-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.

Prolactin Increases the Synthesis of 7α-Hydroxypregnenolone, a Key Factor for Induction of Locomotor Activity, in Breeding Male Newts

Sodefrin is a female-attracting pheromone isolated from the abdominal glands of male newts, Cynops pyrrhogaster. Previously, the preference of conspecific female newts for sodefrin was shown to be completely abolished by plugging the bilateral nostrils, indicating that it acts on the olfactory organ. To determine the sensitivity of the olfactory receptor cells to sodefrin, electro-olfactograms (EOGs) in response to sodefrin solution were recorded from the ventral nasal epithelium of sexually developed female newts. Sodefrin elicited marked EOG responses in a dose-dependent manner on the epithelium of the lateral nasal sinus (LNS) region, a putative vomeronasal organ. In ovariectomized females, treatment with prolactin (PRL) and estrogen markedly enhanced the EOG response to sodefrin. The EOG response to the pheromone was also enhanced considerably by treatment with either PRL or estrogen alone. A slight but significant elevation was observed in castrated males receiving PRL plus estrogen or estrogen alone. It was concluded that the main site of action of sodefrin resides in the lateral sinus region and that sensitivity to sodefrin is under the control of PRL and estrogen. The presence of a sex difference in olfactory sensitivity to the hormones and/or pheromone was also suggested.

Hormonal influence on the olfactory response to a female-attracting pheromone, sodefrin, in the newt, Cynops pyrrhogaster☆

It is now established that the brain has the capability of synthesizing biologically active steroids, termed neurosteroids, that participate in the regulation of various neurophysiological and behavioral processes. However, the neuronal mechanisms regulating the activity of neurosteroid-producing cells have not yet been elucidated. We recently found that, in the frog brain, three enzymes involved in steroid biosynthesis are actively expressed in hypothalamic neurons: 3β-hydroxysteroid dehydrogenase/△5-△4 isomerase (3β-HSD), cytochrome P450 17α-hydroxylase/C17,20-lyase (P450C17) and hydroxysteroid sulfotransferase (HST). Concurrently, we showed that frog hypothalamic explants can convert tritiated pregnenolone ([3H]△5P) into various bioactive steroids, including 17- hydroxypregnenolone (17OH-△5P), progesterone (P), 17-hydroxyprogesterone (17OH-P), dehydroepiandrosterone (DHEA), △5P sulfate (△5PS) and DHEA sulfate (DHEAS). The hypothalamic nuclei, where the 3β-HSD-, P450C17- and HST-expressing neurons are located, receive afferent fibers containing a variety of neurotransmitters and neuropeptides. Here, we show that GABA, endozepines and neuropeptide Y (NPY) regulate neurosteroid biosynthesis.

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K. Yamamoto

Papers

ANNUAL CHANGES OF PLASMA PROLACTIN LEVELS IN THE NEWT, CYNOPS PYRRHOGASTER : Endocrinology