Adaptation to fresh water in the sea snake Hydrophis cyanocinctus: Tissue electrolytes and peripheral corticosteroids

Groups of mature sea snakes, Hydrophis cyanocinctus, were transferred from seawater to tanks of running fresh water and sampled at 10, 16, 21, 27, 31, 35, and 51 days after transfer. The following were measured as parameters of adaptation to fresh water and compared with control (seawater maintained) animals: rate of weight loss; muscle water and ion content; blood hematocrit; plasma osmolarity; plasma Na⁺, K⁺, Cl^?, and urea concentrations. Peripheral levels of corticosterone and aldosterone were measured using competitive protein binding assay and radioimmunoassay, respectively. Adrenal in vitro 18-oxygenase activity was determined radiometrically using [³H]corticosterone. The rate of weight loss was lower in snakes in fresh water, but hematocrit and tissue water composition were unaltered. Plasma ion concentrations and total osmolarity fell significantly within 10 days and stabilized after approximately 16 days. Only after 51 days did muscle ionic composition or plasma $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ alter. Urea was below detectable levels in all animals. Peripheral aldosterone concentrations remained at 1 to 4 μg/100 ml plasma whereas mean corticosterone titers fell significantly from 8 to 3 μg/100 ml. Adrenal in vitro 18-oxygenase activity was, however, significantly increased after 51 days of freshwater treatment. It was concluded that H. cyanocinctus is an osmoconforming euryhaline reptile, and possible roles for the adrenal in hydromineral balance are discussed.     

The pituitary--adrenal axis in the sea snake, Hydrophis cyanocinctus daudin.

The sea snake Hydrophis cyanocintus was subjected to conventional pharmacological tests of pituitary-adrenal function. Administration of synthetic ACTH resulted in adrenal hypertrophy, enhanced Δ⁵-3β-hydroxysteroid dehydrogenase (3β-OHSD) activity and raised peripheral titres of corticosterone and aldosterone to 300% those of controls. Metopirone treatment resulted in adrenal hyperplasia and enhanced 3β-OHSD activity, but aldosterone titres were unchanged despite a 90% reduction in corticosterone titre. Dexamethasone administration was followed by adrenal atrophy and diminished 3β-OHSD activity. Titres of both corticosteroids were much lowered. Despite such marked variations in adrenal function, no alteration in plasma ion concentrations or total osmolarity could be detected. These and earlier data suggest that separate regulatory mechanisms for corticosterone and aldosterone may exist in the sea snake, and such a differential regulation may be of physiological importance.     

Calcitonin content in the ultimobranchial gland of the snake,Elaphe climacophora: Comparison of adults,young, and hatchlings

Hypocalcemic activity of the ultimobranchial gland of Elaphe climacophora was determined using the rat bioassay. The activity of one ultimobranchial gland of the adult females corresponds to that of 55 mU (MRC) of salmon calcitonin. The activities of one gland of the young and the hatchling are comparable to those of 17.5 and 18.0 mU of salmon calcitonin, respectively. The activity per unit body weight is extremely high (2,700 mU/kg body wt.) in the young and the hatchlings in comparison with that in the adults (460 mU/kg body wt.).     

The effects of prolactin and TSH on thyroid function in Fundulus heteroclitus

Treatment with ovine prolactin significantly reduces serum T₄ in Fundulus heteroclitus, both in control (P < 0.05) and TSH-stimulated fish (P < 0.01), without affecting thyroidal radioiodine uptake. Prolactin also reduces the loss of radioiodine from F. heteroclitus (P < 0.001). This loss is markedly increased by ovine TSH (P < 0.001), but only in the absence of prolactin. Although other explanations are possible, prolactin is probably goiterogenic in F. heteroclitus, lowering thyroidal secretion of T₄ as well as inhibiting the peripheral effects of this hormone.     

Bioassay for salmon prolactin using hypophysectomized Fundulus heteroclitus

A bioassay for salmon prolactin (PRL) is described. This assay which is based on the sodium-retaining action of PRL in the hypophysectomized killifish, Fundulus heteroclitus, has proved to be rapid, sensitive (250 pg PRL per gram of fish), and specific. The procedure has been used to characterize the biological activity of a highly purified PRL from the pituitaries of the chinook salmon, Oncorhynchus tschawytscha, and a similar PRL isolated (by acid buffer polyacrylamide disc gel electrophoresis) from pituitaries of coho salmon (O. kisutch) (MW ca. 22,000; isoelectric point >9).     

Correlations between integument structure and ecdysteroid titers in fifth-stage nymphs of the tick, Ornithodoros moubata (Murray, 1877; sensu Walton, 1962)

The integument structure of fifth-stage nymphs of the tick Ornithodoros moubata was studied during the molting processes which are induced by the blood meal. In parallel, the ecdysteroid titers in hemolymph and in extracts from whole animals were determined by a radioimmunoassay (RIA). Between Days 2 and 3 after the meal (a.m.) a few procuticle lamellae were deposited and the mitotic period initiated. Ecdysteroid titers were low during the first 3 days a.m. They began to increase between Days 3 and 4 a.m. with the mitotic period ending. The titers rose sharply when apolysis and formation of the exuvial space took place (Day 4–5 a.m.). Highest ecdysteroid concentrations were measured during the deposition of the new epicuticle (Day 5–6 a.m.). Concomitant with the beginning of procuticle deposition and digestion of the old cuticle (Day 6 a.m.) the titers began to drop and reached low values again shortly before ecdysis (Day 9–10 a.m.). A preliminary chemical identification by thin-layer and high-performance liquid chromatography revealed that the majority of the RIA-positive ecdysteroids were composed of ecdysone and mainly ecdysterone. This was confirmed by gas chromatography of the silylated ecdysteroids. The results demonstrate that the titer changes in relation to the molting events in both ticks and insects appear to be very similar. It is thus likely that ecdysteroids are important in the control of tick molting.     

Fine structure of the corpuscles of stannius of the trout, Salmo gairdneri: Structural changes in response to increased environmental salinity and calcium ions

These studies confirm that there are two cell types, C₁ and C₂, in the corpuscles of Stannius (CS) of the trout, Salmo gairdneri, both of which have the appearance of protein-or peptide-secreting cells. C₁ cells have large secretory granules, up to 1.0 μm in diameter, while the C₂ cells have small granules up to 0.5 μm. In seawater (SW)-adapted trout the C₂ cell granules are significantly larger than those in freshwater (FW)-adapted trout. In fresh water the C₁ cells are relatively inactive and the C₂ cells are mainly sparsely granulated and actively secreting. The converse situation is found in seawater-adapted trout in which the C₁ cells are very active and the C₂ cells are relatively inactive and contain stored granules. During adaptation to SW from FW some C₁ cells develop extensive rough endoplasmic reticulum and Golgi but other C₁ cells degenerate and disintegrate apparently releasing the entire cell contents, including granules, into the capillary lumena. Protein may be released from these granules in capillaries by digestive enzymes from lysosome-like vesicles. In both adapted FW and SW corpuscles the active cells appear to release their granules by exocytosis. The appearance of C₁ cells of fish transferred from FW to calcium-enriched fresh water is similar to that of fish transferred to dilute SW, suggesting that these cells respond to high levels of external calcium, while the C₂ cells are active in media of low ionic and osmotic strength.     

The effects of hypophysectomy and replacement therapy with prolactin,cortisone, or their combination on the blood of the black bullhead Ictalurus melas

Experiments are done on intact and hypophysectimized Ictalurus melas maintained in approximately isotonic saline. Serum Na⁺ decreases after hypophysectomy but serum Cl^? is not affected. Prolactin increases both serum Na⁺ and Cl^? in intact fish, but only Na⁺ increases in hypophysectomized recipients. Cortisone decreases serum Na⁺ in hypophysectomized fish but increases serum Cl^?. When prolactin and cortisone are administered together, the Na⁺-increasing effect of prolactin is dominant, while the Cl^?-increasing effect of cortisone is greatly enhanced. It is concluded that the two ions are regulated separately and that both hormones are necessary to maintain normal levels in hypophysectomized recipients. Serum Ca²⁺ is higher in adult females than in males and low in juveniles, irrespective of treatment. Serum inorganic P is lower in juveniles than in adults and is unaffected by hypophysectomy. Serum organic P increases in hypophysectomized fish receiving cortisone, perhaps in correlation with the increase in serum proteins. Saline injections are hyperglycemic in intact fish but prolactin injections are hypoglycemic, as is hypophysectomy. Serum cholesterol tends to increase after hypophysectomy and injections of prolactin and/or cortisone result in a significant decrease. No sexual differences in serum proteins are detected in adult, postspawning fish and hypophysectomy has no effect in September, but low levels characterize winter fish at low temperature. Cortisone increases serum proteins in hypophysectomized fish in winter, but otherwise no hormonal effects are observed. Prolactin significantly lowers the pH of the blood, alone or in combination with cortisone.     

Effects of two gonadotropic hormones on polysaccharide synthesis in the albumen gland of Lymnaea stagnalis, studied with the organ culture technique

A medium for culturing organs of the basommatophoran freshwater snail Lymnaea stagnalis is described. One of the accessory sex glands, the albumen gland, which produces proteins and galactogen as nutritive substances for the embryos, was cultured along with parts of the central nervous system (CNS), including the female gonadotropic hormone-producing dorsal bodies (DB). Culture time was 4 days. The cerebral ganglia and the DB appeared to have a strong stimulating influence on polysaccharide synthesis in the albumen gland. The remaining part of the CNS has a weak stimulating effect. The use of extracts (culture time 6–26 hr) of parts of the cerebral ganglia and of the DB showed that not only the dorsal body hormone (DBH), but also the ovulation hormone (CDCH)—a neurohormone produced by the caudodorsal cells located in the cerebral ganglia—stimulates the synthetic activity of the albumen gland. The action of these hormones is direct; i.e., it is not exerted via the gonad. Dose- and time-response relations for the DBH and the CDCH are very similar. No additional rise in stimulation occurred when the two hormones were administered together. Experiments with albumen glands of adult snails demonstrated that the presence of large quantities of secretory material in the gland inhibits the response to DBH and CDCH.     

An immunocytochemical investigation for arginine vasotocin and neurophysin in the pituitary gland and the caudal neurosecretory system of Catostomus commersoni.

An immunoperoxidase technique has been used for the localization and identification of arginine vasotocin (AVT) and neurophysin in two neurosecretory systems of the freshwater teleost Catostomus commersoni. In the pituitary gland AVT was found only in the pars nervosa, in close association with neurophysin. In the caudal neurosecretory system, both AVT and neurophysin appeared to be absent.     

The effects of cortisol treatment on carbohydrate and protein metabolism in Fundulus heteroclitus

In male Fundulus heteroclitus captured during the fall, 5 daily injections of cortisol (200 μg/fish or approximately 20 μg/g body wt) produced significant elevations in serum cortisol and hyperglycemia in both fed and fasted fish. Only fasted fish responded with an increase in liver glycogen. No consistent changes attributable to hormone injection occurred in serum protein, amino acid, liver protein, liver amino acids, or liver alanine aminotrasferase. A single cortisol injection (20 μg/g body wt) in fasted fish produced elevated blood glucose levels which persisted for 48 hr and followed serum cortisol changes closely. Again, no significant changes were seen in liver glycogen or in protein metabolism. We conclude that in F. heteroclitus, blood glucose is the major carbohydrate reservoir influenced by cortisol elevation. Protein catabolism appears not to be the source of the glucose elevation. Reduced peripheral glucose utilization and gluconeogenesis from lactate or glycerol are suggested as alternative sources.     

Changes in plasma corticosteroids during smoltification of coho salmon,Oncorhynchus kisutch

Plasma corticosteroid concentrations were determined by competitive protein binding assay in coho salmon, Oncorhynchus kisutch, during smoltification. Between January and April plasma corticosteroid levels decreased from 8.6 to 3.5 ng/ml, but increased to 24.0 ng/ml by the end of May in smolts retained in fresh water. This nearly eightfold increase in corticosteroids occurred as plasma thyroxin levels declined. Thus plasma corticosteroids appear to be increasing during the period when coho salmon normally migrate to sea.     

Immunocytochemical localization of thyroglobulin in the transforming endostyle of anadromous sea lampreys, Petromyzon marinus L., during metamorphosis

Light and electron microscopic immunocytochemistry was used to detect the localization of thyroglobulin (TG) in the transforming endostyle of the anadromous sea lampreys, Petromyzon marinus L., during metamorphosis. TG is found within the transforming endostyle throughout metamorphosis (stages 1 to 7) and in early stages (1–2) staining for TG is similar to that observed in the endostyle of the ammocoete. By stage 3, most of the TG is found in large bodies that accumulate within type 3 cells. At stage 4 immunostaining for TG is found within the epithelial cells of the transforming endostyle and in the lumina of some of the first formed follicles. Most of the follicular lumina contain TG by the end of metamorphosis (stage 7). TG in the young parasitic adult lamprey is localized within the follicular lumina, colloid droplets, and occasionally in large bodies found in some of the follicular epithelial cells.     

The influence of cortisol on the ACTH-producing cells in the pituitary gland of the chick embryo: An immunocytochemical study

The response of 11-day-old chick embryos to exogenous cortisol is explored by the injection of eggs with 1, 10, 50, 100, and 1000 ng of cortisol. Pituitaries, collected 1 hr after each concentration of cortisol and 48 hr after injection of 1000 ng of cortisol, are processed for paraffin embedding and sectioned at 7 μm. ACTH-producing cells are identified with the immunoperoxidase technique employing rabbit antisera against bovine ACTH. Immunoreactive ACTH (Ir-ACTH) positive cells are restricted to the cephalic lobe of the pars distalis and corresponded with the chromophobes of Ehrlich's hematoxylin and eosin stain. Low doses of cortisol from 1 to 100 ng result in an increase accumulation of ACTH within the ACTH cells and also an increase in the numbers of Ir-ACTH-positive cells. At higher concentrations of cortisol (1000 ng), cells are totally depleted of their ACTH granules within 1 hr and are not identifiable with the immunoperoxidase technique. The ACTH cells 48 hr later, however, recuperate showing ACTH-positive granules within the cytoplasmic matrix. It is concluded that cortisol is an active hormone in chick embryos that influences the ACTH cell population via the classical negative feedback mechanisms.     

The abdominal perisympathetic neurohemal organs of the cockroach Periplaneta americana: Innervation revealed by cobalt chloride diffusion

By retrogradely diffusing Co²⁺ through the two perisympathetic organs associated with the transverse nerve of the third abdominal ganglion and subsequently precipitating the sulfide, four groups of neuron somata in that ganglion and two somata in the second abdominal ganglion are revealed. The four groups in the third abdominal ganglion occur at various levels adjacent to the midsaggital plane, and there are 8 to 16 neurons in each group. Comparable numbers of putative neurosecretory cells, with granules that are paraldehyde fuchsin and azocarmine positive, are located in most of these regions. Axons containing granules with these staining characteristics are also present in the perisympathetic organs. It is suggested that the granules are secretory products of the median neurons. The locations of the cell bodies, their sizes, and some of the intraganglionic pathways of their processes are described.     

Synchronous maturation of the female reproductive system and of the ovulation hormone-producing system in Lymnaea stagnalis

The intercerebral commissures (COMs) of young snails, which are in the male stage (10–15 mm shell height), contain only small quantities of ovulation hormone (CDCH): 0.6 OIU · COM^?1 · g^?1 snail (1 ovulation inducing unit = threshold dose for ovulation in adult recipients). During the subsequent period of female maturation the quantities of CDCH increase considerably up to a maximum of 47 OIU · COM^?1 · g^?1 snail at the onset of oviposition. In snails smaller than ca. 21 mm shell height oviposition cannot be induced by COM extract injections. It is concluded that maturation of the female reproductive system and of the ovulation hormone system occur simultaneously, and that both processes are stimulated by the hormone of the dorsal bodies (endocrine organs located upon the cerebral ganglia).     

The influence of exogenous ACTH on the levels of corticosterone and cortisol in the plasma of young chicks (Gallus domesticus)

Ovarian tissues from murrel (Channa punctatus Bloch) were incubated in vitro to obtain discrete effect regarding free cholesterol (f-cholesterol) depletion in response to salmon gonadotropin (SG-G100). To select the best responsive ovarian stage, ovarian tissues containing 30–40% or 40–50% or 60–70% or 80–90% of mature oocytes were incubated separately with SG-G100 (400 ng/incubation). Maximum response was evident in tissue containing 40–50% matured oocytes. With increasing concentrations of SG-G100 like 1.5 and 10 μg/incubation, a distinct linear profile for ovarian f-cholesterol depletion was observed. The linear slope of f-cholesterol depletion was checked for individual variations and striking uniformity and parallelism was observed among the different individuals of murrel. Carp and murrel pituitary extracts were tested in the present system and gonadotropin content was expressed as nanograms of SG-G100 equivalent. Efficacy of fish hypothalamic extract (HE) and gonadotropin releasing hormone (GnRH) was also judged by coincubating ovary and pituitary. Findings clearly indicate that the present in vitro system is not only highly sensitive as a bioassay of teleost gonadotropin but also very convenient since the whole procedure can be done within 10 hr.     

Primary structures of multiple forms of urotensin II in the urophysis of the carp, Cyprinus carpio

Multiple forms of urotensin II (UII), one of the hormonal peptides of the caudal neurosecretory system of fishes, were purified from the urophyses of the carp, Cyprinus carpio. Three distinct peaks with UII activity (classified as UII-α, -β and -γ) were separated by reverse-phase high-pressure liquid chromatography (HPLC). Edman degradations as well as digestion with carboxypeptidase A revealed the primary structures of these peptides as

The results of thin-layer chromatography, HPLC, amino acid analysis, and sequencing indicate that UII-α and -γ are homogeneous. UII-β appears, however, to be a mixture of two components, differing only at position 2. Thus, in the carp urophysis, four forms of UII appear to be present, although the separation of two components in UII-β has not been obtained. Sequence of positions 6–11 is common to all forms of UII isolated from the carp, sucker (Catostomus commersoni) and goby (Gillichthys mirabilis).     

Endocrine regulation of ovarian development in the autogenous mosquito, Aedes atropalpus

The mosquito Aedes atropalpus is autogenous, i.e., normal ovarian development proceeds in adult females without any exogenous source of protein. Using a radioimmunoassay (RIA) for ecdysone, it was found that the titer of antigen-positive material in whole-body extracts of adult females rose from about 150 pg equiv of β-ecdysone per female at eclosion to an average peak value of 323 pg 32 hr later. The titer then dropped to approximately 100 pg over the next 16 hr. A comparison of the RIA titer with follicle size, vitellin deposition into oocytes, and total extractable soluble proteins from ovaries suggested a causal relationship between the hormone titer and normal ovarian development. Preliminary data also suggest that the ovary is a source of ecdysteroid in these adult animals. Decapitation of adult females from 0 to 4 hr after eclosion completely prevented ovarian development. Animals decapitated 30 min after eclosion and then assayed for ecdysteroid content 32 hr later revealed a hormonal titer 75% less than normal intact controls. An analysis of the 32-hr RIA-positive material by high-pressure liquid chromatography confirmed the presence of β-ecdysone but did not detect any α-ecdysone. Ovarian deficiencies incurred by decapitation were partially restored by injection with β-ecdysone or by topical application of juvenile hormone.     

Effect of urophysial homogenates on plasma ion levels in Gillichthys mirabilis (Teleostei: Gobiidae)

In seawater-acclimated Gillichthys mirabilis, 2 and 4 hr after receiving an intraperitoneal injection of Gillichthys urophysial homogenate (two urophyses per fish), plasma sodium, magnesium, and chloride levels were significantly greater than those observed following a saline injection. When sodium and magnesium levels were measured in injected urophysectomized fish, similar increases were noted. No differences were observed in plasma potassium or calcium or in hematocrit between fish receiving saline and those receiving the urophysial homogenate. No differences were observed between urophysectomized and sham-operated fish in plasma sodium, potassium, calcium, and magnesium or in hematocrit 1, 3, 7, or 14 days postoperatively. Transection of the spinal cord at the level of the seventh or eighth preterminal vertebra had no effect on plasma sodium, potassium, calcium, or magnesium compared with sham-operated fish 14 days postoperatively. These findings demonstrate an effect of urophysial factor(s) on osmotic and ionic regulatory mechanisms in a euryhaline marine teleost.