Search

The effects of newt motilin on the contractility of the isolated gastrointestinal (GI) tract from Japanese fire belly newts (newt) were examined to clarify whether motilin regulates GI motility in urodele amphibians. In addition, contractile responsiveness to motilins from seven species of vertebrates (human, chicken, turtle, alligator, axolotol, newt and zebrafish) were compared in GI preparations from three different animals (rabbit duodenum, chicken ileum and newt stomach) to determine the species-specific action of motilin. Newt motilin (10

Motilin, a peptide hormone consisting of 22 amino acid residues, was identified in the duodenum of pigs in the 1970s. It is known to induce gastrointestinal contractions during the interdigestive state in mammals. Although the motilin gene has been identified in various animal species, it has not been studied in amphibians. Here, we identified the motilin gene in the Japanese fire bellied newt (Cynops pyrrhogaster), and conducted an analysis of tissue distribution, morphological observations, and physiological experiments. The deduced mature newt motilin comprises 22 amino acid residues, like in mammals and birds. The C-terminus of the newt motilin showed high homology with motilin from other species compared to the N-terminus region, which is considered the bioactive site. Motilin mRNA expression in newts was abundant in the upper small intestine, with notably high motilin mRNA expression found in the pancreas. Motilin-producing cells were found in the mucosal layer of the upper small intestine and existed as two cell types: open-and closed-type cells. Motilin-producing cells in the pancreas were also found to produce insulin but not glucagon. Newt motilin stimulated gastric contractions but not in other parts of the intestines in vitro, and motilin-induced gastric contraction was significantly inhibited by treatment with atropine, a muscarinic acetylcholine receptor antagonist. These results indicate that motilin is also present in amphibians, and that its gastrointestinal contractile effects are conserved in mammals, birds, and amphibians. Additionally, we demonstrated for the first time the existence of pancreatic motilin, suggesting that newt motilin has an additional unknown physiological role.

A variety of mechanisms are responsible for enabling an organism to escape a predatory attack, including behavioral changes, alterations in hormone levels, and production and/or secretion of toxins. However, these mechanisms are rarely studied in conjunction with each other. The Rough-skinned Newt ( Taricha granulosa ) is an ideal organism to examine the relationships between these mechanisms because its behavioral displays and toxin secretion during a predator attack are well documented and readily characterized. While we found no direct relationship between antipredator behavior and endogenous levels of corticosterone (CORT), antipredator behavior was inhibited when exogenous CORT and adrenocorticotropic hormone (ACTH) were administered, resulting in high circulating concentrations of CORT, indicating that CORT may play a role in mediating the behavior. There was no correlation between the animal’s toxicity and either CORT or behavior. The results of this study provide evidence that CORT plays an important, yet complex, role in the antipredator response of these amphibians.

Seasonally-breeding amphibians have served as excellent animal models to investigate the biosynthesis and biological actions of neurosteroids. Previous studies have demonstrated that the brain of amphibians possesses key steroidogenic enzymes and produces pregnenolone, a precursor of steroid hormones, and other various neurosteroids. We recently found that the brain of seasonally-breeding newts actively produces 7α-hydroxypregnenolone, a previously undescribed amphibian neurosteroid. This novel amphibian neurosteroid acts as a neuronal modulator to stimulate locomotor activity in newts. Subsequently, the mode of action of 7α-hydroxypregnenolone has been demonstrated in the newt brain. 7α-Hydroxypregnenolone stimulates locomotor activity through activation of the dopaminergic system. To understand the functional significance of 7α-hydroxypregnenolone in the regulation of locomotor activity, diurnal and seasonal changes in synthesis of 7α-hydroxypregnenolone have also been demonstrated in the newt brain. Melatonin derived from the pineal gland and eyes regulates 7α-hydroxypregnenolone synthesis in the brain, thus inducing diurnal locomotor changes. Prolactin, an adenohypophyseal hormone, regulates 7α-hydroxypregnenolone synthesis in the brain, and also induces seasonal locomotor changes. In addition, 7α-hydroxypregnenolone mediates corticosterone action to increase locomotor activity under stress. This review summarizes the discovery, progress and prospect of 7α-hydroxypregnenolone, a new key regulator of amphibian locomotion.

It is becoming clear that the vertebrate brain has the capability of forming steroids de novo , the so-called “neurosteroids”. To understand neurosteroidogenesis in the brain, it is essential to demonstrate the formation of pregnenolone, a main precursor of neurosteroids. In amphibians, the pregnenolone formation from cholesterol is still unclear, although the brain accumulates pregnenolone, pregnenolone sulfate and 7α-hydroxypregnenolone. This study was addressed to obtain basic information about pregnenolone formation in the newt brain. Firstly, we demonstrated that the newt brain produces pregnenolone from cholesterol. Subsequently, cDNA encoding cytochrome P450 side-chain cleavage enzyme (P450scc), a key steroidogenic enzyme catalyzing pregnenolone formation, was isolated from the newt. The sequence analysis showed that the isolated P450scc cDNA contained a putative coding region consisting of 1569 bp, which encoded 523 amino acids. The steroid- and heme-binding domains of P450scc were highly shared in amino acids among vertebrates. RT-PCR analysis amplified the authentic fragment corresponding to newt P450scc showed its transcription in the brain. However, the transcription level in the brain was lower than those of the gonad and the kidney including adrenals. The restricted cells in the four major regions of the newt brain, such as the telencephalon, diencephalon, mesencephalon, and rhombencephalon, were demonstrated to express P450scc transcripts by RT-PCR and in situ hybridization. Taken together, these results indicate that the newt brain expresses P450scc mRNA and produces pregnenolone from cholesterol.

Estrogens are essential for normal reproductive activity in both males and females as well as for ovarian differentiation during a critical developmental stage in most vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in amphibians, we isolated cDNAs encoding the estrogen receptors (ERα and ERβ) from the Japanese firebelly newt (Cynops pyrrhogaster), Tokyo salamander (Hynobius tokyoensis), axolotl (Ambystoma mexicanum), and Raucous toad (Bufo rangeri). Full-length amphibian ER cDNAs were obtained using 5′ and 3′ rapid amplification of cDNA ends. The predicted amino acid sequences of these amphibian ERs showed a high degree of amino acid sequence identity (over 70%) to each other. We analyzed the relationships of these amphibian ER sequences to other vertebrate ER sequences by constructing a phylogenetic tree. We verified that these were bona fide estrogen receptors using receptor dependent reporter gene assays. We analyzed the effects of natural estrogens, ethinylestradiol, and DDT and its metabolites on the transactivation of the four amphibian species listed above, and Xenopus tropicalis ERs and found that there were species-specific differences in the sensitivity of these ERs to hormones and environmental chemicals. These findings will expand our knowledge of endocrine-disrupting events in amphibians.

Three types of cDNA encoding the arginine vasotocin (AVT) receptors from the newt, Cynops pyrrhogaster were cloned and the gene expression of each receptor analyzed in the organs and tissues of the newt. The deduced amino acid sequence of one type of AVT receptor, consisting of 418 amino acid residues, showed a high degree of sequence identity with the mammalian arginine vasopressin (AVP) V1a receptors (61-68%). The second type of cDNA, encoding an amino acid sequence consisting of 367 amino acid residues, exhibited a relatively high sequence identity with mammalian AVP V2 receptors (50-51%). The third cDNA, encoding a sequence of 415 amino acid residues, possessed high sequence identity with mammalian AVP V3/V1b receptors (59-63%). Phylogenetic analysis revealed that the first, second and third types of receptor were close to mammalian AVP V1a, V2 and V3/V1b receptors, respectively, and RT-PCR using gene specific primers for each type of receptor indicated that the first and second types of receptor mRNA were expressed in various organs and tissues, including the circulatory, osmoregulatory, and reproductive organs of both male and female newts. In contrast, mRNA expression of the third cDNA was mainly detected in the brain and pituitary, and its expression pattern was distinctly different from that of the other two. We suggest that the first, second and third types of newt AVT receptor obtained in the present study are counterparts of mammalian AVP V1a, V2 and V3/V1b receptors, respectively.

Male plethodontid salamanders produce courtship pheromones that increase female receptivity. Three protein components of the courtship pheromone cocktail have been characterized in the mental gland of Plethodon shermani, the red-legged salamander: plethodontid receptivity factor (PRF), plethodontid modulating factor (PMF), and sodefrin precursor-like factor (SPF). In this study, a streamlined in situ hybridization (ISH) protocol, employing a biotinylated oligonucleotide probe, is used to visualize the sites of pheromone expression in formalin-fixed paraffin-embedded P. shermani mental gland and post-cloacal tail tissue. Results corroborate previous RT-PCR studies on pheromone expression. PRF and PMF are highly expressed in P. shermani mental gland, while SPF expression is more variable. None of the tested pheromones is expressed in dorsal or ventral tail glands. The reported protocol is simple, rapid, and effective, allowing visualization of high-copy mRNA transcript in formalin-fixed tissue.

Ghrelin has been identified in some amphibians and is known to stimulate growth hormone release and food intake as seen in mammals. Ghrelin regulates gastrointestinal motility in mammals and birds. The aim of this study was to determine whether ghrelin affects gastrointestinal smooth muscle contractility in bullfrogs (anuran) and Japanese fire belly newts (urodelian) in vitro. Neither bullfrog ghrelin nor rat ghrelin affected longitudinal smooth muscle contractility of gastrointestinal strips from the bullfrog. Expression of growth hormone secretagogue receptor 1a (GHS-R1a) mRNA was confirmed in the bullfrog gastrointestinal tract, and the expression level in the gastric mucosa was lower than that in the intestinal mucosa. In contrast, some gastrointestinal peptides, including substance P, neurotensin and motilin, and the muscarinic receptor agonist carbachol showed marked contraction, indicating normality of the smooth muscle preparations. Similar results were obtained in another amphibian, the Japanese fire belly newt. Newt ghrelin and rat ghrelin did not cause any contraction in gastrointestinal longitudinal muscle, whereas substance P and carbachol were effective causing contraction. In conclusion, ghrelin does not affect contractility of the gastrointestinal smooth muscle in anuran and urodelian amphibians, similar to results for rainbow trout and goldfish (fish) but different from results for rats and chickens. The results suggest diversity of ghrelin actions on the gastrointestinal tract across animals. This study also showed for the first time that motilin induces gastrointestinal contraction in amphibians.

Reproductive behavior in amphibians, as in other vertebrate animals, is under the control of multiple hormonal substances. Prolactin (PRL), arginine vasotocin (AVT), androgen, and 7α-hydroxypregnenolone (7α-OH PREG), four such substances with hormonal activity, are known to be involved in the expression of the tail vibration behavior which is the initial step of courtship performed by the male newt, Cynops pyrrhogaster. As current information on the interaction(s) between these hormones in terms of eliciting tail vibration behavior is limited, we have investigated whether the decline of expression of tail vibration behavior due to suppression of the activity of any one of these hormones can be restored by supplying any one of the other three hormones exogenously. Expression of the behavior was determined in terms of incidence (% of test animals exhibiting the behavior) and frequency (number of times that the behavior was repeated during the test period). Neither PRL nor androgen restored the decline in the incidence and frequency of the tail vibration behavior caused by the suppression of the activity of any one of other three hormones. AVT completely restored both the anti-PRL antibody-induced and flutamide (an androgen receptor antagonist)-induced, but not ketoconazole (an inhibitor of the steroidogenic CYP enzymes)-induced decline in the incidence and frequency of the tail vibration behavior. The neurosteroid, 7α-OH PREG, failed to restore flutamide-induced decline in the incidence and frequency of the behavior. However, it was able to restore both anti-PRL antibody-induced and AVT receptor antagonist-induced decline in the incidence, but not in the frequency of the behavior. In another experiment designed to see the activity of hormones enhancing the frequency of the tail vibration behavior, AVT was revealed to be more potent than 7α-OH PREG. The role of each hormonal substance in determining the expression of the tail vibration behavior was discussed based on the results.

Two full-length cDNAs, encoding delta (delta) and mu (mu) opioid receptors, were cloned from the brain of the rough-skinned newt Taricha granulosa, complementing previous work from our laboratory describing the cloning of newt brain kappa (kappa) and ORL1 opioid receptors. The newt delta receptor shares 82% amino acid sequence identity with a frog delta receptor and lower (68-70%) identity with orthologous receptors cloned from mammals and zebrafish. The newt mu receptor shares 79% sequence identity with a frog mu receptor, 72% identity with mammalian mu receptors, and 66-69% identity with mu receptors cloned from teleost fishes. Membranes isolated from COS-7 cells transiently expressing the newt delta receptor possessed a single, high-affinity (Kd = 2.4 nM) binding site for the nonselective opioid antagonist [3H]naloxone. In competition binding assays, the newt delta receptor displayed highest affinity for Met-enkephalin, relatively low affinity for Leu-enkephalin, beta-endorphin, and [D-penicillamine, D-penicillamine] enkephalin (DPDPE) (a delta-selective agonist in mammals), and very low affinity for mu-, kappa-, or ORL1-selective agonists. COS-7 cells expressing the newt mu receptor also possessed a high-affinity (Kd = 0.44 nM) naloxone-binding site that showed highest affinity for beta-endorphin, moderate-to-low affinity for Met-enkephalin and Leu-enkephalin and DAMGO (a mu-selective agonist in mammals), and very low affinity for DPDPE and kappa- or ORL1-selective agonists. COS-7 cells expressing either receptor type (delta or mu) showed very high affinity (Kd = 0.1-0.3 nM) for the nonselective opioid antagonist diprenorphine. Taricha granulosa expresses the same four subtypes (delta, mu, kappa, and ORL1) of opioid receptors found in other vertebrate classes, but ligand selectivity appears less stringent in the newt than has been documented in mammals.

Seasonal changes in LHbeta and FSHbeta mRNA levels were examined in the pituitary gland of the adult male newt, Cynops pyrrhogaster, using in situ hybridization histochemistry and a quantitative real-time RT-PCR method. The annual fluctuation of LHbeta mRNA and FSHbeta mRNA levels in the pituitary gland displayed a close relationship with seasonal changes in testicular function. The values obtained by both methods showed similar fluctuation. The levels of LHbeta mRNA were always exceeded those of FSHbeta. The present immunoelectron microscopic observations support the data on the gene expression levels of the beta-subunits of LH and FSH. Gonadectomy in the summer increased the LHbeta and FSHbeta mRNA levels. Testosterone replacement inhibited the expression of LHbeta mRNA, but not of FSHbeta mRNA, suggesting that the expression of FSHbeta is regulated by some non-steroid factor, probably inhibin. In the case of gonadectomy during any other season, the LHbeta mRNA level increased, but not to the same extent as in summer, and androgen concentrations decreased to the minimum of the year. This finding provides new information about the regulation of annual changes in LHbeta and FSHbeta expression in the pituitary gonadotrophs.

The effects of intracerebroventricular (ICV) and intraperitoneal (IP) injections of ovine prolactin (PRL), antiserum against newt PRL, and antibody against the newt PRL receptor on the expression of courtship behavior of male newts, Cynops pyrrhogaster, were studied to see whether PRL acts centrally or peripherally to induce the behavior. Injections of PRL by either route into gonadotropin-primed males enhanced the expression of the behavior dose-dependently. The minimum effective amount of ovine PRL administered intracerebroventricularly was 0.1 microg, whereas it was 100 microg when injected intraperitoneally. ICV injection of antiserum against newt PRL blocked the spontaneously occurring male courtship behavior when the anti-newt PRL serum was given either intracerebroventricularly or intraperitoneally. The minimum effective dose of the antiserum administered intracerebroventricularly was 0.05 microl, whereas it was 20 microl when injected intraperitoneally. Neither ICV nor IP injection of preimmune serum affected the expression of the behavior. Furthermore, ICV, but not IP, administration of 0.3 microg of anti-newt PRL receptor antibody, purified from antiserum against newt PRL receptor by use of an antigen-conjugated affinity column, blocked the spontaneously occurring courtship behavior in sexually developed males. Neither ICV nor IP injection of the same amount of normal rabbit IgG affected the expression of the behavior. The results strongly suggest that endogenous PRL enhances the behavior by acting centrally through the PRL receptors localized in the brain area.

Environmental temperature plays important roles for amphibian gonadal function. In this study, we examined the responses of testicular tissue of adult male newts (Cynops pyrrhogaster) to amphibian gonadotropins both in vitro and in vivo under different temperatures. When minced testes were incubated in vitro at different temperatures (8-37 degrees C) under an atmosphere of 95% O(2)-5% CO(2) for 3h with bullfrog luteinizing hormone (LH) or follicle-stimulating hormone (FSH), LH stimulated testosterone production more than FSH. The testosterone production increased as the incubation temperature increased. Hypophysectomized newts were injected with bullfrog LH or FSH and maintained at 8 or 18 degrees C. In the 18 degrees C group, the testicular weight of the hypophysectomized control decreased when compared with that of with the intact control. The testicular weight of the LH-treated hypophysectomized group decreased more than that of the hypophysectomized control, indicating that LH induced the evacuation of mature spermatozoa from the testes of LH-treated hypophysectomized newts. In the FSH-treated newts, the testicular weight was greater than that in the hypophysectomized control, and was maintained at a value similar to that of the intact control. In the 8 degrees C group, there was no significant difference in testicular size among the intact control, hypophysectomized control, and FSH-treated newts. LH strongly induced spermiation as it did at 18 degrees C. The plasma testosterone level in the hypophysectomized newts decreased dramatically, but LH was effective in restoring it. Its effect was more potent at 8 degrees C than at 18 degrees C. On the other hand, FSH did not induce a significant increase in the plasma testosterone levels at either temperatures. The results indicate a temperature-dependent difference in responsiveness of the testis both in vitro and in vivo to LH and FSH.

In newts elevated titers of plasma prolactin (PRL), induced by low temperature, cause apoptosis in the penultimatemitotic stage of spermatogonia, and this cell death is suppressed by antiserum against newt PRL, but only during the initial 3 days of exposure (Yazawa et al., 1999). Thus, factors other than PRL must be involved in spermatogonial death. Follicle-stimulating hormone (FSH) may be a plausible candidate. Accordingly, the current study examined the activityof FSH on the proliferation and survival of spermatogonia at low temperatures in vivo and in vitro. Porcine FSH (pFSH) administration in vivo inhibited spermatogonial death induced at 12 degrees C, but failed to do so at8 degrees C. Also pFSH promoted in vitro the proliferation of spermatogonia at 12 degrees C, but not at 8 degrees C. Furthermore,dibutyryl cyclic AMP stimulated in vitro DNA synthesis of secondary spermatogonia at 12 degrees C, but not at 8 degrees C. These different responses to temperatures were not caused by different levels of mRNA for the receptor of follicle-stimulating hormone, the numberof FSH binding sites, or FSH binding affinity to its receptors in the testicular cells. Thus, the results indicate that a temperature-sensitive period exists duringthe postreceptor process and is responsible for thelack of response of newt testis to FSH at 8 degrees C.

A partial prolactin (PRL) cDNA was specifically PCR amplified from a cDNA library constructed from pituitary mRNAs of the newt (Cynops pyrrhogaster) and cloned into plasmid vectors. One clone thus obtained contained a 739-bp insert encoding the C-terminal amino acid sequence of the mature hormone molecule. Using this clone as a probe, the full-length newt PRL cDNA was screened from the cDNA library. The PRL cDNA clone thus obtained consisted of 1024 bp encoding the entire sequence of the mature PRL molecule in addition to its signal peptide. The amino acid sequence of newt PRL deduced from its nucleotide sequence showed higher homologies with those PRL sequences of tetrapod animals than with those of teleosts. Northern blot analysis revealed the newt PRL mRNA size to be approximately 1 kb. In situ hybridization using the newt PRL cDNA as a probe revealed that the pituitary region expressing PRL mRNA corresponded to that immunoreactive with antiserum against PRL. PRL mRNA levels in the pituitary of newts subjected to room and low temperatures were determined by Northern analysis employing the PRL cDNA as a probe. PRL mRNA levels were significantly higher in the pituitaries of newts subjected to 10 degrees than in those of newts kept at 23 degrees. Likewise, immunoassayable plasma PRL levels were higher in animals subjected to 10 degrees than in those kept at 23 degrees.

We have isolated cDNA clones encoding molecules of putative glycoprotein hormone alpha subunit precursors from their pituitary cDNA libraries for a toad, bullfrog and newt. The insert of the isolated toad cDNA was 562 bp long containing the 5′-untranlated, coding and 3′-untranslated regions of 38, 363 and 161 bp, respectively. The insert of the bullfrog cDNA was 604 bp long containing the 5′-untranslated, coding and 3′-untranslated regions of 70, 366, and 168 bp, respectively. In the newt, a composite cDNA sequence was estimated from four isolated partial clones. It was 694 bp long and contained the 5′-untranslated region of 89 bp, coding region of 366 bp, and 3′-untranslated region of 91 bp or longer. Amino acid sequences deduced from coding regions of the isolated clones showed that the signal peptides consist of 24 residues and the mature proteins of 96 (toad) or 97 residues (bullfrog and newt). In all three species, an insertion of an amino acid residue was found between residues 26 and 27 of the alpha subunit molecule sequence of all other vertebrate species studied. Interestingly, the percentage identities of the entire amino acid sequence between amphibian and mammalian (or avian) alpha subunits are lower than those between lungfish and mammalian (or avian) alpha subunits. This suggests that amino acid substitutions have occurred more frequently during the course of evolution in the alpha subunit molecule of amphibians than in that of other tetrapod vertebrates, although the biological significance of this is not known.

The cDNA sequences encoding the mesotocin receptor (MTR) and vasotocin 1a receptor (VTR-1a) were identified in a urodele amphibian, the rough-skinned newt, Taricha granulosa . Saturation binding of [ 3 H]oxytocin (OT) to the Taricha MTR (tMTR) was best fit by a two-state model; a high affinity-low abundance site and a lower affinity-high abundance site. Competition-binding studies found the following rank-order affinities for the tMTR: mesotocin (MT) > OT ≈ vasotocin (VT) > vasopressin (VP) > isotocin (IT). Inositol phosphate (IP) accumulation studies demonstrated functional activity of both the tMTR and Taricha VTR-1a (tVTR-1a) in a heterologous cell culture system. The rank-order potencies for the tMTR were MT > OT > VT ≈ VP > IT. The combined binding and IP results indicate that VT may act as a partial agonist of the tMTR. Rank-order potencies for the tVTR-1a were VT > VP > MT ≈ OT > IT. For both receptors, stimulation of IP accumulation was blocked by d(CH 2 ) 5 [Tyr(Me) 2 ]AVP (Manning compound) and d(CH 2 ) 5 [Tyr(Me) 2 ,Thr 4 ,Tyr-NH 2 ]OVT (OTA). OTA was a more potent antagonist for the transiently expressed tMTR while Manning compound was relatively more potent at inhibiting IP accumulation in tVTR-1a expressing cells. In contradiction to earlier assumptions, the absolute IC 50 of Manning compound was lower for the tMTR (27 nM ± 13) than the tVTR-1a (586 nM ± 166) indicating its potential higher affinity for the tMTR, a finding with special relevance to interpretation of comparative studies investigating the behavioral and physiological actions of neurohypophysial peptides in non-mammalian species.

We now know that steroids can be synthesized de novo by the brain and the peripheral nervous system. Such steroids are called neurosteroids and de novo neurosteroidogenesis from cholesterol is a conserved property of vertebrate brains. Our studies over the past decade have demonstrated that the brain expresses several kinds of steroidogenic enzymes and produces a variety of neurosteroids in sub-mammalian species. However, neurosteroid biosynthetic pathways in amphibians, as well as other vertebrates may still not be fully mapped. We first found that the newt brain actively produces 7alpha-hydroxypregnenolone, a previously undescribed amphibian neurosteroid. We then demonstrated that 7alpha-hydroxypregnenolone acts as a novel bioactive neurosteroid to stimulate locomotor activity of newt by means of the dopaminergic system. Subsequently, we analyzed the physiological roles of 7alpha-hydroxypregnenolone in the regulation of locomotor activity of newt. This paper summarizes the advances made in our understanding of 7alpha-hydroxypregnenolone, a newly discovered bioactive amphibian neurosteroid stimulating locomotor activity, and its physiological roles in the regulation of locomotion in newt.

The effects of purified prolactins isolated from frogs (fPRL; Rana catesbeiana) and newts (nPRL; Cynops pyrrhogaster) were compared with those of ovine prolactin (oPRL) and thyrotropin releasing hormone (TRH) in the eft (Notophthalmus viridescens) skin transepithelial potential (TEP) bioassay. At total doses as low as 0.4 micrograms/animal, both fPRL and nPRL were as effective as oPRL in reducing eft skin TEP. By contrast, TRH at total doses as high as 400 micrograms/animal was ineffective in altering eft skin TEP. These data provide the first direct evidence that amphibian PRLs can exert significant physiological control over salamandrid integumental sodium transport.

Antiserum against bullfrog growth hormone (fGH) was produced by immunizing rabbits with the highly purified fGH obtained from adenohypophyses of adult bullfrogs. Histological studies on bullfrog adenohypophyses revealed that the cells that immunologically reacted with the antiserum against fGH corresponded to the ones positively stained with the antiserum against rat GH. The antiserum together with fGH and 125I-fGH was employed to develop a radioimmunoassay (RIA) for fGH. Several dilutions of plasma and of pituitary homogenate of both adult and larval bullfrogs yielded dose-response curves which were parallel to the standard curve. Ovine prolactin (PRL), and growth hormone (GH); eel and salmon GHs; and bullfrog LH, FSH, TSH, PRL, and neurointermediate lobe homogenate did not react in this assay. Plasma from hypophysectomized bullfrogs had no detectable immunoreactive GH. Pituitary homogenates of Bufo japonicus, Xenopus laevis, and Cynops pyrrhogaster gave inhibition curves which did not parallel the standard. The homologous RIA for bullfrog GH thus developed was applied for the determination of plasma and pituitary GH levels in the larvae and adults. Plasma GH levels were relatively low during preclimax period and rose as metamorphosis progressed. Plasma GH concentrations were maximum in the juvenile frogs and decreased as the animals grew up. Pituitary GH concentrations also increased as metamorphosis progressed. After metamorphosis, pituitary GH concentrations declined as the frogs gained weight. There was no sex difference in plasma and pituitary GH levels in the adult.

Courtship-induced changes in plasma steroid and brain gonadotropin-releasing hormone (GnRH) concentrations in Taricha granulosa were determined with respect to changes in female sexual receptivity. Females were sacrificed at several times after courtship initiation. Concentrations of GnRH (determined by RIA) in the anterior telencephalon were high at courtship initiation (females unreceptive), but decreased by sperm transfer (females receptive). Courtship had no affect on GnRH concentrations in any other brain region examined. Furthermore, courted, receptive females had higher plasma levels of estradiol than did uncourted controls, and estradiol levels remained elevated above control levels 24 hr after courtship initiation. Courtship had no influence on plasma progesterone or corticosterone levels. To determine if the observed changes in GnRH concentrations in the telencephalon were localized to the nervus terminalis, courted females and controls were sacrificed after 5, 20, or 60 min of courtship. Nervus terminalis GnRH concentrations were higher in courted females than in uncourted controls. These results may represent the first documentation of a naturally occurring physiological change in the nervus terminalis.

We amplified and identified, for the first time in urodele amphibians, cDNA sequences that encode preprovasotocin (preproVT) and prepromesotocin (preproMT) from two distinct urodelian species, Taricha granulosa (the rough-skinned newt) and Plethodon shermanii (the spotted salamander). Each of these cDNA sequences encoded proteins that contained the characteristics of known neurohypophysial peptide precursors; each sequence consisting of (1) a signal peptide, (2) VT- or MT-like peptides, (3) neurophysin, and for the preproVTs, (4) copeptin. In T. granulosa, cDNA sequences encoded for the nine amino acids that define VT or MT. In P. shermani, cDNA sequences encoded for the VT peptide and a previously unidentified isoform of MT, ([Val(4)]-MT).

We have previously shown FSH promotes spermatogonial proliferation and their differentiation into primary spermatocytes in organ culture of newt testicular fragments. Several growth factors identified in newt testis, such as stem cell factor (SCF), insulin-like growth factor (IGF)-I, and neuregulin (NRG)1, also stimulate spermatogonial proliferation in the organ culture. However, any growth factor added in vitro might not work on spermatogonia directly, but act on somatic cells such as Sertoli cells or pericystic cells, because size-selective barrier exists around a cyst which is enclosed by Sertoli cells. In order to determine the target somatic cells of the growth factors as well as the role of pericystic cells in spermatogonial proliferation and differentiation, we searched for agents that kill pericystic cells selectively. We found that treatment of the testicular fragments with trypan blue (TB) caused cell death of only pericystic cells and significant abolishment of the activity of SCF to stimulate spermatogonial proliferation, while the activities of neither IGF-I nor NRG1 were affected. In addition, the potency of neither IGF-I nor FSH to stimulate the differentiation of spermatogonia into primary spermatocytes was abolished by TB treatment. Consistent with these results, only the mRNA expression of c-kit was reduced by TB treatment, whereas those of FSH receptor, SCF, IGF-I, IGF-I receptor, Immunoglobulin-like domain-containing NRG1, ErbB2, and ErbB4 were unaffected. These results indicate that SCF stimulates pericystic cells, while IGF-I and NRG1, as well as FSH, activate Sertoli cells, resulting in stimulation of spermatogonial proliferation in organ culture of testicular fragments.

A large cDNA fragment that codes for proenkephalin (PENK) was cloned from the rough-skinned newt, Taricha granulosa (GenBank Accession: AY817670). This 1299-bp PENK cDNA extends from the poly(A) sequence on the 3' end into the 5'-UTR (221bp) upstream of an open reading frame that codes for 264 amino acids and a stop codon. Within the precursor are five Met-enkephalin sequences and two C-terminally extended forms of Met-enkephalin (YGGFMRGV and YGGFMRY). The organization of the opioid core sequences within the newt PENK closely resembles that reported for other vertebrates. In this urodele amphibian, as in anurans, PENK does not contain the penultimate Leu-enkephalin opioid sequence found in mammals, and instead has in this position Met-enkephalin. PENK cDNA was amplified from newt brain in a RACE PCR targeting the 3' end of the newt delta opioid receptor (DOR). It remains to be determined whether generating the cDNA for the newt PENK while cloning its receptor was serendipitous or the result of a meaningful coincidence between the DOR and PENK sequences.

Vasotocin (VT) and vasopressin control many endocrine and neuroendocrine functions, including the regulation of reproductive behaviors. In the roughskin newt (Taricha granulosa), VT administration can enhance courtship behaviors in males and egg-laying behaviors in females. This study used immunohistochemistry to investigate whether there are sex differences in VT in specific brain areas, and whether these differences persist in nonbreeding animals. Numbers of VT immunoreactive (ir) cell bodies were counted in males and females collected in February, April, June, and August. Radioimmunoassay of plasma samples confirmed that testosterone and 5alpha-dihydrotestosterone concentrations were higher in males than females, and that 17beta-estradiol concentrations were higher in females than males. In 11 brain areas, no sexual or seasonal differences in the number of VTir cells were found. But in 3 brain regions-the bed nucleus of the stria terminalis (BNST), the nucleus amygdalae dorsolateralis (AMYG), and the anterior preoptic area (aPOA)-there were significantly greater numbers of VTir cells in males than in females, and these differences did not change seasonally. In the aPOA, an area important to male sex behaviors, the sexual dimorphism in VTir was particularly pronounced. In four brain regions, there were significantly greater numbers of VTir cells in females than males, but only in specific seasons. In April-collected (breeding) animals, more VTir cells were found in females than in males in the populations of VT cells within the pars dorsalis hypothalami and ventromedial hypothalamus, brain regions frequently associated with stress responses and female mating behaviors. In August-collected (nonbreeding) animals, more VTir cells were found in females than in males, in the region of the bed nucleus of the decussation of the fasciculus lateralis telencephali and in the nucleus visceralis superior, nucleus isthmi region. Significantly greater numbers of VTir cells were observed in the magnocellular preoptic area of males and females collected in February. These results indicate that the functional interactions between gonadal steroid hormones and VT are complex and appear to involve site-, sex-, and season-specific regulatory mechanisms. Furthermore, it seems likely that populations of VT neurons in the BNST, AMYG, and aPOA are involved in regulating male-specific behaviors, and that the VT neurons in the pars dorsalis hypothalami/ventromedial hypothalamus may be involved in female-specific behaviors.

Complementary DNA (cDNA) encoding newt (Cynops pyrrhogaster) growth hormone (nGH) was cloned from a cDNA library constructed from mRNAs of newt pituitary glands and was expressed in Escherichia coli. Based on Northern blot analysis using the cDNA as a probe, the nGH mRNA was estimated to be 940 bases in length. The recombinant nGH (nGHr) had a molecular mass of 22 kDa as determined by SDS-PAGE and possessed considerable bioactivity as determined in a Xenopus cartilage assay. Using the nGHr, we produced a polyclonal antibody against nGHr. Western blot analysis of newt anterior pituitary gland homogenates revealed that this antiserum specifically detected a single 22-kDa band, and histological studies of newt pituitary gland sections showed that the cells that reacted immunologically by the anti-nGHr antiserum corresponded to those stained by an antiserum against rat GH. A radioimmunoassay (RIA) that is specific and sensitive for nGH was developed, employing the antiserum thus produced. The sensitivity of the RIA was 57 +/- 7 pg/100 microl assay buffer. Interassay and intraassay coefficients of variation were 1.22 and 2.70%, respectively. Serial dilutions of plasma and pituitary homogenate of C. pyrrhogaster yielded dose-response curves that were parallel to the standard curve. Plasma from hypophysectomized newts showed no cross-reactivity. Moreover, displacement curves obtained using pituitary homogenates of the sword-tailed newt (C. ensicauda) and the crested newt (Triturus carnifex) were also parallel to the standard curve. Mammalian and frog GHs and prolactins (PRLs), as well as newt PRL, showed no inhibition of binding, even at relatively high doses, in this RIA. The RIA was used to measure GH released from newt pituitaries in vitro. Enhancement of GH release by 10(-7) M thyrotropin-releasing hormone was observed in cultures of newt pituitaries.

A specific and sensitive radioimmunoassay (RIA) was developed for the measurement of sodefrin, a female-attracting decapeptide pheromone of the red-bellied newt, Cynops pyrrhogaster. Sodefrin synthesized according to the amino acid sequence of native sodefrin, isolated from the abdominal glands of the cloaca of the male newt, was used as a reference standard. An antiserum to sodefrin was produced by immunizing a rabbit with synthetic sodefrin that was extended on its C-terminus with Cys coupled to hemocyanin. For the radioligand, sodefrin N-terminally extended with Tyr was used. An aqueous extract of the abdominal glands of C. pyrrhogaster produced a displacement curve parallel to the sodefrin standard, whereas that from the sword-tailed newt (Cynops ensicauda) showed no inhibition of binding in this RIA. The sensitivity of the RIA was 30.5 +/- 3.4 pg/100 microliters assay buffer. Intraassay and interassay coefficients of variation were 1.6 and 4.5%, respectively. The RIA was used to determine sodefrin levels in the abdominal gland of the male newt. Hypophysectomy and castration greatly reduced the sodefrin content. Administration of testosterone propionate (TP) to the hypophysectomized and castrated newt increased the pheromone content in the abdominal gland. A combination of prolactin (PRL) and TP elevated the sodefrin content markedly, while PRL alone scarcely affected it. Immunoreactive sodefrin was observed in the epithelial cells of the abdominal gland of both hormone-treated and saline-injected newts. Among them, groups treated with PRL plus TP and TP alone exhibited strong immunoreactivity in their abdominal gland compared with PRL-treated and saline-injected groups.

In order to ascertain the involvement of endogenous prolactin (PRL) in the expression of courtship behavior by the male newt, Cynops pyrrhogaster, antiserum against homologous PRL was administered to male newts, which had been exhibiting courtship behavior in the field and had been taken to the laboratory. The effect of the antiserum on the preference of terrestrial over aquatic habitats also was investigated. Within 24 hr of the first injection of antiserum, both incidence and frequency of courtship behavior declined markedly compared with those in the preimmune serum-injected newts. The anti-PRL serum-induced decline became more conspicuous thereafter. These animals came to prefer a terrestrial habitat. Cessation of antiserum injection and administration of PRL restored the expression of courtship behavior to a certain extent and altered their habitat preference from terrestrial to aquatic. The results strongly suggest the involvement of endogenous PRL in the expression of courtship behavior as well as in the preference of aquatic habitat.

For understanding the evolutionary relationships between gonadotropins [GTHs: lutropin (LH) and follitropin (FSH)] and their receptors, we attempted to characterize the extracellular domain of the receptors, which is thought to be a key region of hormone binding, in nonmammalian species, and to compare the information to that of the known mammalian data. For this purpose, we designed two sets of sense and antisense oligonucleotides as polymerase chain reaction (PCR) primers, referring to the known mammalian GTH receptors, such as LH receptors of human, pig, and rat, and FSH receptors of human and rat. All possible combinations of the primers showed the successful amplification of cDNA of LH receptor without contamination of FSH receptor cDNA from rat testicular RNA samples. With these primers, reverse transcription (RT)-PCR was applied to the gonads of nonmammalian species (quail, snake, tortoise, newt, and bullfrog). Only the quail, however, showed the specific amplification when only one set of primers was used. Thus, the PCR product of the quail was used as a probe for Northern blot and in situ hybridization. By Northern blot analysis, a single size of mRNA (3 kb) was identified from quail testicular poly A + RNA. The distribution of mRNA visualized by in situ hybridization was limited only on Leydig cells of quail testis. These results suggest that a part of the quail LH receptor cDNA was amplified by RT-PCR. The nucleotide and predicted peptide sequences of this amplified cDNA were compared with those of mammalian receptors. The size of characterized cDNA sequence was 519 bp, which is completely identical with those of mammalian LH receptors. The homology of both cDNA and predicted peptide was about 70% of those of mammalian LH receptors (intramammalian, about 80%). In spite of the relatively low homology, the positions of cystein residues and potential N-linked glycosylation sites in the peptide were completely conserved in all species compared (human, pig, rat, and quail). The conserved potations indicate their importance for the molecular conformation and specific ligand binding activity of LH receptors.

Changes in plasma concentrations of androgens, estradiol-17β, and progesterone were examined throughout the year in Hynobius nigrescens. The highest mean concentration of androgens (312.7 ng/ml) was found in males in the process of spermiation and having just entered the breeding pond in early spring. Plasma estradiol- 17β concentration was at its base level in females both before and after the animals had entered the pond. Plasma progesterone concentrations were low throughout the year except during the aquatic phase in March when females possessed some ova within the coelom and many ova in the oviducts and ovisacs after the completion of ovulation. During the terrestrial phase, or nonbreeding season, plasma concentrations of androgens and estradiol-17β were in approximate correspondence with the development of sex accessory structures. This is the first documentation of plasma concentrations of sex steroids in hynobiid salamanders.

In the newt, Cynops pyrrhogaster, parathyroidectomy (PTX) brought about a marked decrease in the concentration of plasma calcium. The animals recovered from the hypocalcemia by 15 days after the operation if the pituitary gland was left intact. After PTX, no significant changes in the plasma sodium concentration were observed. Experiments were then conducted to obtain direct evidence that endogenous prolactin (PRL) is involved in this recovery process. Recovery of the calcium level after PTX was blocked by administration of an antiserum raised against newt PRL. In newts deprived of both the pituitary and parathyroid glands, no recovery from hypocalcemia was observed. Administration of newt or ovine PRL to parathyroidectomized-hypophysectomized newts significantly elevated the blood calcium level. After PTX, the concentration of immunoassayable PRL in the blood rose to 10 times the value in sham-operated animals. These results indicate the involvement of PRL in calcium homeostasis in newts with a shortage of parathormone.

A specific and sensitive homologous radioimmunoassay (RIA) for newt ( Cynops pyrrhogaster ) prolactin (PRL) was developed. PRL isolated fromnewt pituitary glands was used for generating antiserum in a rabbit, for radioiodination, and for the standard. Several dilutions of plasma and pituitary homogenate of newts yielded dose-response curves which were parallel to the standard curve. Plasma from hypophysectomized newts showed the least amount of cross-reaction. Pituitary homogenates of other species of urodeles such as least amount of cross-reaction. Pituitary homogenates of other species of urodeles such as Ambystoma mexicanum and Onychodactylus japonicus gave inhibition curves which were parallel to the standard curve. Purified PRLs of anurans such as Rana catesbeiana and Bufo japonicus gave inhibition curves which did not parallel the standard. Bovine PRL and ovine PRL showed no inhibition of binding even at relatively high doses in this RIA. The RIA was applied to the determination of plasma and pituitary PRL levels in the adult newts treated with dopamine agonist (bromocriptine) and/or antagonist (pimozide). Pimozide enhanced PRL levels and bromocriptine antagonized it, while pituitary PRL levels were not appreciably changed.

The distribution and characteristics of putative arginine vasotocin (AVT) receptors in the urodele amphibian kidney were investigated using in vitro quantitative autoradiography. Specific binding sites for [3H]arginine vasopressin (AVP) in the kidney of the rough-skinned newt (Taricha granulosa) were located over the glomeruli. Scatchard analysis showed that, in the range of concentrations tested (0.2 to 22 nM), [3H]AVP bound to a single class of receptors with a dissociation constant of 1.4 nM and a binding site concentration of 36.5 fmol/mg protein. Binding displacement studies showed that both AVT and AVP were potent ligands for newt kidney receptors. Two specific antagonist peptides with anti-vasopressor (V1) activity, but not anti-antidiuretic (V2) activity, in rat tissues were tested as well. Both antagonists effectively displaced [3H]AVP from receptor sites in newt kidney slices, indicating that the binding sites in this amphibian resemble the V1 subtype of mammals in ligand specificity. Localization of AVT receptors over kidney glomeruli and ligand specificity of these sites is consistent with the hypothesis that AVT may cause antidiuresis in urodele amphibians at least in part via a glomerular vasoconstricting action.

Luteinizing hormone-releasing hormone (LHRH) concentrations were measured in specific brain areas of male rough-skinned newts collected from a single population throughout the reproductive cycle. Plasma androgen and corticosterone (B) concentrations were also measured. Androgen concentrations were highest during the breeding season (winter) and lowest during the summer. In contrast, plasma B was lowest during the breeding season and highest in the summer. Concentrations of LHRH in the infundibulum (I) and rostral hypothalamus (RH) were positively correlated throughout the reproductive cycle; LHRH was always higher in the I than in the RH. Concentrations of LHRH in the ventral preoptic area (POA) fluctuated independently of concentrations of LHRH in the I and RH. However, LHRH concentrations decreased to undetectable levels in all three brain areas in March and April, which was before the end of the breeding season and before plasma androgen concentrations had decreased. An injection of LHRH into postbreeding males resulted in a significant increase in plasma androgen concentrations, indicating that the pituitary-gonad axis was still functional at the end of the breeding season. These results support the hypothesis that an abrupt decline in LHRH secretion is the initial endocrine event that signals the end of the breeding season in this amphibian.

In the crested newt ( Triturus cristatus carnifex Laur.), osmotic permeability and active sodium transport (the latter studied by the short-circuit current technique) in the isolated skin change cyclically during the year. The lowest values for both parameters are recorded in winter (aquatic phase), while the highest values are recorded in summer (terrestrial phase). Prolactin (24 IU of ovine prolactin subdivided into three injections delivered on alternate days) administered during the terrestrial phase (coincident with the low rate of hormone release) induces a drastic drop in both osmotic permeability and active sodium uptake. Conversely, hypophysectomy performed during the aquatic phase increases both the osmotic permeability and active transport of sodium across the skin. The role played by the skin in the osmoregulatory mechanism of the crested newt is discussed.

Thyroid glands from red-spotted newts were cultured in medium containing ovine thyroid stimulating hormone (o-TSH), ovine prolactin (o-PRL), or o-TSH and o-PRL in combination. In other preparations thyroid glands were maintained with pituitary glands in cultures to which various concentrations of thyrotropic regulating hormone (TRH) had been added. By radioimmunoassay, concentrations of thyroxine (T4) were measured in samples of medium take after various periods of culture. No effect of TRH on the release of T4 was detected. Levels of T4 were raised by TSH but not by PRL. Greater increases were induced by TSH + PRL than by TSH alone. It is concluded that under the conditions of this study PRL enhances the effect of TSH on the release of T4 by the thyroid gland in the red-spotted newt.

The testis of the urodele amphibian Taricha granulosa was studied with regard to localization of androgen production and stimulation of androgen secretion by mammalian LH. Plasma testosterone (T) and 5α-dihydrotestosterone (DHT) increased in proportion to the amount of ovine LH injected; this stimulatory effect of ovine LH was essentially abolished by bilateral castration. In vitro androgen secretion from testicular fragments of the mature zone far exceeded that from fragments of the immature zone. These data indicate that in urodeles plasma T and DHT are produced by cells predominantly localized in the mature zone of the testis, even before spermiation has occurred.

Immunoreactive neurons were seen in the preoptic area and in the tuberal region of the hypothalamus of the newt. Somatostatin immunopositive fibers were present throughout the hypothalamus and formed a massive innervation of the median eminence, both in median and lateral areas, while the pars nervosa was devoid of reaction. The pattern of somatostatin immunoreactive systems was different from that shown by immunocytochemical methods against neurophysin.

A single pair of parathyroid glands is typical of the red-spotted newt but an auxilary pair is sometimes present. The gland is composed of a single type of epithelial cell arranged in cords. The structure of the gland is stable throughout the year and shows no sexual dimorphism. Parathyroidectomy causes the calcium levels of both the blood and the urine to fall but control values are reestablished within 2 weeks. Administration of mammalian parathyroid extract (PTE) produces a rise in the concentration of calcium in the urine but no change in the calcium of the blood. Plasma phosphate rises after parathyroidectomy but the phosphate level of the urine remains unchanged. Within 24 hr of the injection of PTE a transitory peak in the phosphate appears in both blood and urine. It is suggested that in the red-spotted newt the parathyroid hormone mobilizes calcium from bone but, perhaps, may not act on the kidney.

Ovine prolactin has been shown to initiate porphyropsin synthesis in “second metamorphosis” of the eastern red-spotted newt ( Notophthalmus viridescens ). Prolactin activity is demonstrable in both terrestrial juvenile efts and aquatic adults. In adults, thyroxine initiates molting, antagonizes prolactin-induced water-drive behavior, and yet does not appear to affect visual pigment synthesis. Adaptation of adults to aquatic and terrestrial conditions does not alter visual pigment composition; prolactin initiates porphyropsin synthesis in animals maintained in both experimental environments.

Integumental transepithelial potential (TEP) was measured by both in vivo and in vitro (Ussing chamber) techniques in the same adult terrestrial-phase California newts (Taricha torosa). In both types of preparation, TEP showed a logarithmic relation to external sodium (as Na2SO4) concentrations between 0.1 and 10 meq/liter, with in vivo values exceeding in vitro values at all points. KM was ca. 1 and 5 meq/liter for the in vivo and in vitro preparations, respectively. When terrestrial-phase newts were treated with prolactin (PRL; 50 mIU/g/day), in vivo TEP declined significantly within 3 days. In vivo TEP versus [Na+]ext curves of recently collected aquatic-phase newts were found to be not significantly different from those of PRL-treated terrestrial-phase animals. Current/voltage (I/V) plots likewise showed that skin from aquatic-phase and PRL-treated terrestrial-phase newts was electrophysiologically alike. Estimates and calculations based on the I/V relationship suggest that PRL acts primarily to increase the epithelial electrical resistance in the transcellular pathway, with no effect on the electromotive force of the sodium pump.

Thyrotropins (TSH) and gonadotropins (luteinizing hormone, LH, and follicle-stimulating hormone, FSH) purified from ovine, bovine, ostrich, sea turtle, and bullfrog pituitary glands were tested for their ability to simulate the thyroid glands of four species of amphibians (three anurans and a urodele), a reptile (the slider turtle), and a bird (the cockerel). All animals tested responded specifically to mammalian, sea turtle, and ostrich thyrotropins; any thyroid-stimulating activity of the gonadotropins from these species could be accounted for by their contamination with thyrotropin. In addition, the three anuran species showed a high degree of specificity for bullfrog TSH; bullfrog LH exhibited low thyrotropic activity in amphibians. In contrast, bullfrog LH was more potent than bullfrog TSH in stimulating the thyroid gland of the cockerel. This bird therefore resembles reptiles in showing a “heterothyrotropic” response to bullfrog LH. The hormonal specificity of thyroid response to these pituitary glycoprotein hormones therefore varies distinctly among representatives of different vertebrate classes. Although the tetrapods tested were generally specific for thyrotropin in their thyroid responses to homologous and heterologous pituitary hormones, occasionally a lack of specificity for heterologous hormones existed which could be shared by species from closely-related classes. The varied patterns seen indicate that independent evolution of the functional characteristics of the thryotropin receptor has occurred to different degrees within each vertebrate class.

Steroidogenesis by the ripe ovarian tissue of Triturus alpestris alpestris was studied in vitro using both incubation and superfusion techniques. The following metabolites were identified by their isopolarity and isomorphicity with authentic compounds after the tissue was incubated or superfused with [4- 14 C]progesterone, 11-deoxycorticosterone, 11-deoxycortisol, 17α-hydroxyprogesterone, androstenedione, and testosterone. Product yield versus time curves approached a steady-state plateau in the superfusate and displayed a differently timed single peak in the incubation medium. In both cases, the integrated yields of 11-deoxycorticosterone and, to a minor extent, of 11-deoxycortisol were greater than those of androgens. The amounts of 11-deoxycorticosterone retained by the superfused and incubated tissues also exceeded those of the other identified compounds. No steroid hormone was isolated after incubation with sodium [1- 14 C]acetate, even in the presence of ovine LH or newt pituitary homogenate. On the other hand, 11-deoxycorticosterone was produced from endogenous precursors during superfusion of ovarian tissue with medium alone (0–180 min) or with medium containing HCG (10 IU/ml; 180–300 min). During the first period, 11-deoxycorticosterone output declined to a baseline of 2 ng/g/hr, from which it increased sixfold in the presence of HCG. The final 11-deoxycorticosterone concentration in the tissue was 46.7 ng/g. This steroid was measured by radioimmunoassay following a chromatographic separation from interfering compounds.

The effects of various hormonal conditions on epidermal proliferation were studied in the red-spotted newt. Prolactin was found to be a strong stimulant of epidermal mitotic activity. Hypophysectomy caused a rapid decline in the mean mitotic index, the thickness, and the number of cell layers in the epidermis. These parameters were increased by administration of ovine prolactin in both intact and hypophysectomized newts and by autografting of the pituitary gland. Injection of the prolactin inhibitor, ergocornine, into autografted newts decreased mitotic activity. Thyroid hormone increased the mean mitotic index of intact newts whereas thyroidectomy decreased the mitotic activity. The inhibitory effect of hypophysectomy was not prevented by exogenous thyroxine, indicating that thyroid hormone alone does not stimulate mitosis. Thyroid hormone apparently stimulates mitosis indirectly by increasing the rate at which sloughs are shed. Evidence for this view comes from the observation that the goitrogen thiourea, which mimics thyroidal effects on sloughing, also stimulates mitosis. Conversely, the accumulation of unshed sloughs may, in part, be responsible for the decrease in the mitotic activity in the thyroidectomized newts. Administration of adrenocorticotrophic hormone produced no detected effect on epidermal proliferation.

DNA, RNA, and protein contents, RNA polymerases (I and II), and DNA polymerase activities have been measured in Mullerian ducts from female, immature newts treated with 17β-estradiol. The hormone largely enhances RNA, DNA, and protein synthesis: maximal RNA polymerase activity (both I and II) occurs at the 4th day of treatment with a concomitant RNA content enhancement (maximal at Day 10); DNA polymerase activity is maximal at the 10th day of treatment, just preceding maximal DNA content (12th day); the β-estradiol effect upon protein synthesis occurs much later (protein content still enhances at the 20th day of treatment).

In vivo integumental transepithelial potential (TEP) in the Eastern red-spotted newt (Notophthalmus viridescens) increases linearly with external [Na+] from 0.1 to 10 mM and is anion independent. Both integumental TEP and osmotic permeability are higher in laboratory-conditioned (LC, terrestrial) than in breeding-condition (BC, aquatic) newts at temperatures of 5-25 degrees. Prolactin (PRL) treatment of LC newts decreased both TEP and rate of water uptake. Arginine vasotocin (AVT) treatment resulted in a substantial increase in water uptake in LC newts, while little or no AVT response was seen in PRL-treated or BC newts. Hypophysectomy (HX) or ergocryptine treatment increased TEP in BC newts, whereas HX + PRL maintained TEP at control levels. Although ergocryptine and HX were without effect on water uptake in BC newts kept at 5 degrees for 9 days, HX + ACTH increased water uptake. HX produced a substantial fall in serum [Na+] in BC newts, while either PRL or ACTH replacement elevated serum [Na+]. Combined ACTH and PRL treatment returned serum [Na+] to control levels. These data suggest that high endogenous prolactin plays a significant role in maintaining serum [Na+] and integumental permeability and transport characteristics in breeding-condition N. viridescens. Although PRL and ACTH are both sodium retaining in the aquatic breeding stage, these two hormones promote opposite effects on the integument; PRL decreases both water uptake and integumental TEP, whereas ACTH (presumably acting through the adrenals) increases water uptake and possibly TEP.

Sex steroid levels in the plasma of the male Pleurodeles waltl were determined by radioimmunoassay of blood samples taken monthly from each newt during an 18-month period. Androgen levels varied according to season with annual peaks in October-November and in March. The position of these two peaks was the same in the 2 years studied. Testosterone was the principal androgen measured, while the levels of dihydrotestosterone and 4-androstenedione were lower but fluctuated in parallel with that of testosterone. The 17 beta-estradiol levels were very low or undetectable. Androgen levels during the year were synchronous between the several newts studied. The cyclical variations in the steroid levels agreed with the morphological modifications of sexual characteristics during the annual cycle.