In vivo regulation of steroidogenesis by ovine gonadotropins in male and female mudpuppies,Necturus maculosus Rafinesque

Using steroid radioimmunoassay the in vivo steroidogenic responses of male and female mudpuppies (Necturus maculosus) to single injections of ovine FSH and LH were investigated. In males, the major effect of LH was to stimulate testosterone and 5α-dihydrotestosterone secretion within 2 hr of injection, with a lesser effect on estrogen (estrone and estradiol-17β) secretion. In contrast, FSH primarily stimulated secretion of estrogens. The effects of both FSH and LH on steroid secretion in males were dose related. Castration of males reduced plasma androgen, and estrogen to very low or nondetectable levels and abolished the steroidogenic response to LH. Interrenalectomy prevented a postcastration rise in the progesterone level. In females, FSH did not stimulate steroid secretion but LH increased plasma androgens. Homologous pituitary extracts also stimulated plasma levels of all gonadal steroids measured.     

Involvement of gonadotrophin and steroid hormones in spermiation in the amago salmon, Oncorhynchus rhodurus, and goldfish, Carassius auratus

Effects of intraperitoneal injections of chum salmon gonadotrophin (SGA) and various steroid hormones (17α,20β-dihydroxy-4-pregnen-3-one; 17α,20β-diOHprog, 17α-hydroxy-progesterone, testosterone, 11-ketotestosterone) on the induction of in vivo spermiation were examined in nonspermiating amago salmon (Oncorhynchus rhodurus) and goldfish (Carassius auratus). A single injection of SGA to amago salmon and two successive injections to goldfish induced strong spermiation responses. Similarly two successive injections of 17α, 20β-diOHprog caused precocious spermiation in both species; however, the response to 17α,20β-diOHprog was of lesser magnitude than that to SGA. The spermiation response of goldfish to 17α-hydroxyprogesterone was similar to that of 17α,20β-diOHprog. Neither testosterone nor 11-ketotestosterone were effective in inducing spermiation in amago salmon, but these steroids were found to be slightly effective in goldfish. Effects of a single injection of SGA on changes in serum levels of 17α,20β-diOHprog and 11-ketotestosterone were also examined in nonspermiating amago salmon. Serum levels of 17α,20β-diOHprog dramatically increased after treatment and peaked on Day 2 and thereafter declined quickly. Similarly, 11-ketotestosterone peaked on Day 2, but the levels remained high throughout the experimental period. Considered together, these findings are consistent with the suggestion that 17α,20β-diOHprog is involved in the process of spermiation in teleosts. It is further suggested that this hormone is a testicular steroidal mediator of gonadotrophin-induced spermiation in amago salmon.     

Circulating thyroid hormone concentrations in relation to seasonal events in the male Indian garden lizard, Calotes versicolor

Circulating thyroxine (T4) and triiodothyronine (T3) were estimated by RIA in plasma samples of Calotes versicolor collected over a period of 28 months. Monthly observations on seasonal events, viz., molting, whole body O2 consumption, abdominal fat deposition, and testes weight were also made. Plasma T4 ranged from 0.16 to 4.63 ng/ml and T3 from 0 to 1.76 ng/ml during the course of the year. Peaks of T4 and T3 could be related with prehibernating (October/November) and posthibernating (March/April) periods, when scale shedding and O2 consumption were also enhanced. Maximum abdominal fat deposition was found in prehibernatory months (October/November). Thyroid hormone levels declined sharply with entry into hibernation. Abdominal fat deposits gradually decreased, being almost depleted by the end of hibernation. The gradual increase in thyroid hormone concentration in the later half of hibernation may be associated with increased lipolysis and preparation of the oncoming reproductive period. Although T4 and T3 profiles were apparently parallel, discrepancies were observed. Posthibernatory peak of T4 was maintained much longer than that of T3 which declined earlier. Also the rise in the prehibernatory T3 concentration was delayed as compared to that in T4.     

Age-related changes in the water and electrolyte metabolism in male rats of the Wistar/TW strain

The changes of water intake, urinary volume, ratio of the water intake to the urinary output (water turnover rate) and urinary concentrations of sodium, potassium and chloride ions were investigated in male rats of the inbred Wistar/Tw strain during aging. Marked increase of the water intake, the urinary output and the water turnover rate as well as marked decrease of the urinary electrolyte concentrations were observed at the age of over 16 months. First signs of increase in the water intake and the water turnover rate were observed at the age of 12-13 months, but the initial reduction of urinary electrolyte concentrations was detected as early as at 7-9 months of age.     

Stimulation of interrenal secretion in amphibia. I. Direct effects of electrolyte concentration on steroid release

Using a perifusion system, the direct regulation of isolated interrenal glands of Rana temporaria by electrolyte concentrations was studied. Isolated interrenals reacted for more than 5 hr to mammalian ACTH. Both natural corticosteroids, aldosterone and corticosterone, were released into the perifusion medium in approximately the same amounts. The threshold dose was about 1 mU ACTH given during 5 min. Elevation of Na⁺ concentration decreased the amounts of both corticosteroids released into the medium while elevation of K⁺ increased the amounts. The dose-dependent effect was studied in the range 90–184 mM Na⁺ and 2.8–27 mM K⁺. The two corticosteroids responded somewhat differently to changing electrolyte concentrations but there was insufficient evidence to suggest that the release of the two steroids was regulated differentially. From the time course of steroid release it was clear that production of the hormones was regulated as well as their release.     

Characterization of hepatic growth hormone binding sites in two fish species, Gillichthys mirabilis (Teleostei) and Acipenser transmontanus (Chondrostei)

To obtain information on the presence of growth hormone (GH) receptors in liver of nonmammalian vertebrates the specific binding of 125I-bovine growth hormone (bGH) to liver membranes of seven species representing the major groups was studied by radioreceptor assay. A substantial degree of specific binding was detected with sturgeon (Acipenser transmontanus) liver membranes and a much lower amount was detected on hepatic membranes of Gillichthys mirabilis. No significant specific binding was detected on liver membranes of pigeon, turtle, bullfrog, tilapia, or leopard shark. Gillichthys and sturgeon liver membranes were further characterized and compared with hepatic membranes from male rabbits. The sturgeon and Gillichthys membranes showed binding that was dependent upon time, temperature, pH, and membrane concentration. Scatchard analysis of the binding of 125I-bGH to sturgeon and rabbit membranes revealed both high and low affinity binding sites. The high affinity sites had KA values of 3.1 X 10(11) and 1.0 X 10(11) M-1, and capacities of 12 and 50 fmol/mg protein, respectively. Membranes from Gillichthys liver contained only a single class of binding sites with a KA of 6.7 X 10(9) M-1 and a binding capacity of 49 fmol/mg. Hormonal specificity of the sturgeon and Gillichthys hepatic binding sites was studied using methionyl-human GH (met-hGH), ovine prolactin (oPRL), and a crude preparation of sturgeon (st)GH. The met-hGH and stGH inhibited the binding of 125I-bGH to sturgeon liver membranes while only met-hGH displaced labeled bGH from Gillichthys liver membranes. One microgram of oPRL did not significantly inhibit 125I-bGH binding in either membrane assay. Based on these studies, sturgeon hepatic GH receptors seem to be more like those of nonprimate mammals than those of teleosts. Our results, in conjunction with the data of J. N. Fryer (Gen. Comp. Endocrinol. 39, 123-130 (1979)), indicate that considerable evolutionary divergence has occurred among teleost hepatic GH receptors. Thus, vertebrate GH receptors seem to have undergone at least as much evolution as has the hormone itself.     

Breeding condition, temperature, and the regulation of salt and water by pituitary hormones in the red-spotted newt, Notophthalmus viridescens

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.     

Estrogen synthesis in vitro and in vivo in the brain of a marine teleost (Myoxocephalus)

Conversion of radiolabeled substrate to estrone and estradiol was studied in a marine teleost, Myoxocephalus octodecimspinosus (longhorn sculpin), a species previously shown to have exceptionally high brain aromatase activity. In vitro aromatization of ³H-labeled androstenedione was greatest in homogenates of the preoptic area/anterior hypothalamus, medial telencephalon, and lateral telencephalon, in that order, and when measured in individual fish, marked sex and seasonal differences were observed. Relatively little estrogen was produced by the inferior lobes of the hypothalamus, thalamus, optic lobes, basal midbrain, cerebellum, and medulla. Sculpin testis was aromatase negative, and at no time did ovarian activity per unit weight exceed that in the brain. Addition of 11β-hydroxy- and 11-ketotestosterone to forebrain homogenates did not alter estrogen yields. Following perfusion of a sculpin head preparation with ³H-labeled 19-hydroxyandrostenedione, estrone was isolated from the efferent perfusate and estrone and estradiol recovered from the brain and pituitary. The authenticity of formed estrogen in this experiment was verified by derivative formation and recrystallization. Estrogen concentration was highest in the preoptic area/hypothalamus, somewhat lower in the medial and lateral telencephalon, and lowest in the remaining brain regions. Pituitary estrogen per unit weight was at least 12 times higher than in any brain region and, in contrast to the brain in which estrone predominated, the greatest proportion of total estrogen was estradiol. No authentic estrogen was recovered from non-neural tissues (liver, kidney, heart, muscle, skin, and gills) after perfusion. All of the above findings show that the highest levels of aromatase activity correspond neuroanatomically to the reported distribution of estrogen-binding cells and the location of reproductive control centers in the teleost brain. In these regions, a correlation between CNS estrogen production and reproductive status (gender and season) is further evidence for a functional interrelationship. The presence of relatively large amounts of estrogen in the sculpin pituitary following perfusion with radiolabeled androgen suggests that, at this level of phylogeny, the brain may regulate pituitary function by supplying estrogen directly, or the pituitary itself is capable of aromatization. Whether or not brain-derived estrogen makes a significant contribution to the peripheral estrogen pool is still open to question.     

Corticosteroid receptors in the kidney of chick embryo. III. Nature, properties, and ontogeny of aldosterone receptor

An aldosterone receptor in the cytosol from kidney of chick embryos which had a sedimentation coefficient of 8.2 S and a molecular weight higher than 100,000 was identified. Kinetic analysis at 4 degrees revealed a rapid association of the hormone to the receptor that followed second-order reaction kinetics and a dissociation of pseudo-first-order reaction kinetics. The association (ka) and dissociation (kd) rate constants were, respectively, 4.94 X 10(5) M-1 sec-1 and 8.33 X 10(-6) sec-1. From their ratio a KA value of 5.9 X 10(10) M-1 was calculated. In a series of experiments performed with kidneys of 17-day-old embryos, the KA at equilibrium, obtained from the Scatchard plot, was 3.1 +/- 1.2 X 10(8) M-1, whereas the Nmax was 172 +/- 14 fmol/mg protein. Competition studies with various steroids demonstrated that corticosterone had an affinity for the receptor close to that of aldosterone, thus suggesting a degree of resemblance of the mineralo- and glucocorticoid receptors in the chick embryo. However, the profiles of the binding affinities and capacities during the embryogenesis showed that the aldosterone-binding sites had a pattern completely different from that of the glucocorticoid receptor, indicating that the two receptors are most likely separate entities.     

Ovarian cholesterol dynamics in teleost Channa punctatus (Bloch): Relationship with reproductive cycle and response to gonadotropins

Thin-layer chromatography was employed to separate free and esterified cholesterol from total ovarian cholesterol in the teleost Channa punctatus. Results clearly showed that free cholesterol was almost double the concentration (5.37 ± 0.32 nmol/mg) of esterified cholesterol (2.67 ± 0.26 nmol/mg). An annual profile of free and esterified cholesterol determined per ovary showed that both altered in different months of the year although fluctuation was more prominent with free cholesterol. In contrast, the study in terms of per milligramovarian tissue, showed fluctuation of free cholesterol only. Among the pituitaries collected in different reproductive stages, prespawning pituitary extract had maximum depletive effect on ovarian total and free cholesterol in comparison to others. Ovine LH (1 μg/100 g body wt) could effectively induce the depletion of ovarian free cholesterol; the most remark-able effect was obtained with a similar dose of SG-G100 but there was insignificant alteration of esterified cholesterol. In hypophysectomized fish, SG-G100 in increasing doses (1 to 10 μg/100 g body wt) evoked a linear response in regard to the ovarian free cholesterol depletion. These findings contrast strikingly with reports on the mammalian ovary.     

High temperature and corticosteroid in the lymphocytes of domestic fowl (Gallus domesticus)

Exposure to an environmental temperature of 42° for 1 hr increased the concentration of endogenously produced corticosteroid bound in the cytoplasm and the quantity of corticosteroid in the nucleus of the lymphoid cells of the bursa of Fabricius, thymus, and spleen of young White Rock chickens. When heat episodes were applied 8–12 hr before the final episode, the increases of corticosteroid in the cells were suppressed relative to the number of preliminary heatings. These results demonstrate an in vivo relationship between environmentally induced corticosteroid response and the potential immunoreactive lymphoid cells.     

In vivo estrogen synthesis by the developing chicken (Gallus gallus) embryo

The concentrations of unconjugated and conjugated estrogens in the allantoic and amniotic fluids of chicken embryos have been followed during incubation. The estrogens estrone, estradiol-17β and estradiol-17α were present as their conjugates in the allantoic fluid of female embryos but not of male embryos. No estrogens were detectable in the amniotic fluid of embryos of either sex. Estradiol-17β glucuronide, the most abundant estrogen present in female allantoic fluid, was first detectable (159 pg/ml) at Stage 35 of development increasing in concentration to 4210 pg/ml at Stage 45. The concentration of estrogen in allantoic fluid of the partially decapitated embryos at Day 18 (Stage 41) of incubation was not significantly different from that of the intact stage 41, approximately Day 15 embryo.     

Cortisol and osmotic adjustment of the euryhaline teleost, Sarotherodon mossambicus

Changes of plasma cortisol concentration in the euryhaline Sarotherodon mossambicus were studied during the first time of adaptation to different external salinity conditions. The fish survived for more than 24 hr after direct transfer to saltwater only when the concentration was not higher than 2.7%. Transfer from fresh water directly to 2.7% saltwater increased the plasma cortisol level only during the first 2 hr. Plasma osmolality and sodium level were raised for more than 72 hr. Fish adapted for some weeks to 3.5% saltwater and transferred back to fresh water showed a similar increase of cortisol concentration which lasted for 6 hr. Plasma osmolality and sodium were lowered for a longer time (more than 6 days). Transfer from fresh water to distilled water was followed by an increase of plasma cortisol for 2 days. The significance of the increase of cortisol can be seen in the induction of metabolic changes necessary for volume regulatory processes. To test this hypothesis, the influence of cortisol injections was studied in a second set of experiments. Pretreatment with cortisol before the transfer from fresh water to 2.7% saltwater reduced the shrinkage of the muscle cell and enlarged especially the extracellular space within the muscle. It is evident that shifts of electrolytes are stimulated by cortisol.     

Effects of monosodium l-glutamate on pituitary innervation in goldfish, Carassius auratus

Monosodium l-glutamate was injected intraperitoneally into goldfish at a dosage of 2.5 mg/g body wt. The first noticeable effect in the pituitary, 30 min postinjection, was a marked swelling of pituicytes in the neurohypophysis (NH). Eighteen hours postinjection a large number of degenerating type B fibers were found in the NH of the proximal pars distalis (PPD), while all type A fibers, and type B fibers in other parts of the pituitary, remained unaffected. Because glutamate causes a lesion in the nucleus lateral tuberis (NLT), from the anterior margin of the pituitary stalk through to the posterior end of the nucleus, and a minor lesion in the nucleus preopticus periventricularis, it is likely that the NLT is the origin of the degenerative type B fibers in the proximal NH. The significance of these results is discussed in relation to the neuroendocrine regulation of the pituitary functions in goldfish.     

Subcellular localization and preliminary characterization of islet hormone-degrading activities in anglerfish islet tissue

The degradation of [125I]iodoinsulin in anglerfish islet tissue was studied in a trichloroacetic acid solubilization assay system. The pH optima for insulin breakdown by acidic and neutral enzymes were determined in fish islets and compared with mammalian tissues (rat liver, pancreas, and islets of Langerhans). Two major insulinolytic activities of anglerfish islet tissue were partially characterized: 1) An acidic (pH 3.5) activity showing marked sensitivity to pepstatin, some sensitivity to antipain, leupeptin, phenylmethanesulfonyl fluoride (PMSF), and thiol proteinase inhibitors, but no inhibition by EDTA; and 2) a neutral (pH 7.3) activity showing marked sensitivity to thiol proteinase inhibitors, sensitivity to antipain and leupeptin, but no sensitivity to PMSF, EDTA, or pepstatin. Glucagonolytic activities were also observed at the same acidic and neutral pH optima. Following cell fractionation of anglerfish islet homogenates, acidic (pH 3.5) insulinolytic activities were distributed with the lysosome-rich, microsome, and cytosol fractions, whereas neutral (pH 7.3) activities were found chiefly in cytosol and microsomes. Little or no insulinolysis was observed in secretory granule fractions. The data suggest that insulin is degraded in islet tissue by at least two enzyme systems. Lysosomal insulinolysis was due principally to cathepsin D-like activity. Neutral insulinolysis, partially characterized in the cytosol fraction, was due to thiol proteinase activity. The activity profile indicates that islet tissue resembles other insulin-responsive tissues in its subcellular distribution of insulin-degrading activities. The cellular heterogeneity of islet tissue, and the presence of high concentrations of endogenous islet hormones require further efforts at purification before insulinolytic enzymes are fully characterized in this tissue.     

Effect of the presence of females on the pituitary-testicular activity in male Japanese quail (Coturnix coturnix japonica)

Five experiments were carried out to study the role of the presence of a female on the reproductive endocrinology of male Japanese quail. In the first three experiments, exposure of an adult male raised in long days to a female for 10 min or 1 week failed to increase plasma testosterone and LH levels; in fact a significant transitory decrease in plasma testosterone was observed, associated with a preceding increase in plasma corticosterone. These changes are interpreted as a result of the stress caused by repeated bleeding or by the continuous presence of a female in a limited space. In the last two experiments, an increase in the maturation rate of immature males could be observed in birds maintained in the continuous presence of females by comparison with birds kept in isolation. The paired males had larger cloacal glands and testes and higher plasma levels of testosterone and LH than the isolated ones. This effect of the female was observed in long days (16L:8D) as well as in marginally stimulating short days (12L:12D).     

Effects of urotensin I on the isolated dorsal aorta of the chukar, Alectoris graeca.

The effects of purified urotensin I (UI) from the Catostomus urophysis on arterial blood vessels were examined using isolated dorsal aortic strips from a bird, the chukar, Alectoris graeca. UI at concentrations greater than 1 mU/ml produced dose-dependent relaxation. The same effect was more clearly evident in aortic strips contracted by norepinephrine beforehand. The relaxation following UI was not inhibited by propranolol (10⁻⁶M) or atropine (10⁻⁶M), which markedly reduced the relaxation induced by isoproterenol or acetylcholine, respectively. The dose-dependent contractile response to norepinephrine was not significantly affected by UI, whereas it was competitively blocked by phentolamine (10⁻⁷, 10⁻⁶M). Arginine vasotocin (AVT) at low concentrations (0.1 to 1.0 ng/ml) caused relaxation in 50% of the preparations and slight contraction in the other preparations; at higher concentrations AVT caused contraction in all preparations. In comparison with the response to UI, AVT-induced relaxation developed rapidly. UI at concentrations ranging from 10 to 100 mU/ml also elicited relaxation of the strips contracted by AVT. These results suggest that UI-induced aortic relaxation is not mediated through adrenergic or cholinergic receptors, and that it is unlikely that UI and AVT act by similar mechanisms.     

Hormonal activation of protein kinase and lipid mobilization in the locust fat body in vitro

Cyclic nucleotide-activated protein kinase in locust fat body is elevated by extracts containing adipokinetic hormone (AKH) from locust corpora cardiaca or by synthetic redpigment-concentrating hormone (RPCH) which also exhibits lipid-mobilizing activity. Maximal activation is first attained in vitro 90 sec after RPCH is added to the incubation medium and 3.5 min after addition of AKH extract. This activation reflects the increase in endogenous cyclic nucleotides. Incubation of fat body in the presence of exogenous cyclic nucleotides activates lipase, and RPCH stimulation ultimately elevates the level of transported diacylglycerol 2.3-fold. RPCH stimulates fat body lipase in vivo.     

Corticosteroid receptors in the kidney of chick embryo II. Ontogeny of corticosterone receptor and cellular development

In this study the ontogeny of cytosol receptors for corticosterone in the chick embryo kidney was examined and then this ontogenic profile was compared with that of an index of cellular development, i.e., the enzyme ornithine decarboxylase (ODC). The corticosterone receptor concentration (n_max) increased by sevenfold from Day 12 to Day 15 of embryogenesis and then declined to its lowest level by the time of hatching (Day 21). Similar results were obtained when dexamethasone was used as ligand, except that the baseline values at Day 9 and 21 were higher than those found with corticosterone. An identical ontogenic profile was obtained when the results were corrected for the endogenous glucocorticoids present in the cytosol. This increase of the corticosterone receptor occurred simultaneously with the enhanced adrenal corticoid synthesis. The ODC also showed a marked increase and a rapid fall during chick embryogenesis, but the enzyme activity was at its maximum when the corticosterone receptor number was still low (Days 12 and 13) and quickly decreased by the time the receptors had reached their highest levels (Days 14 and 15). The lowest level of ODC was observed immediately before hatching. These results indicate that during chick embryogenesis adrenal corticoids may induce the development of the corticosteroid receptor and that such development may cause a suppression of ODC activity. This suppressive effect of glucocorticoids could represent a mechanism of hormonal action on the kidney.     

The effects of aldosterone on sodium and potassium metabolism in larval Ambystoma tigrinum

Electrolyte depletion resulting from 14 days in distilled water, which was changed daily, lowered plasma Na concentration from 108 to 92 mM. Aminoglutethimide was administered on each of the final 3 days of the depletion period in doses of 3 mg/animal. This further depressed Na to 75 mM. Both aldosterone (25 micrograms/animal) and corticosterone (200 micrograms/animal) returned plasma Na to 92 mM. Cortisone (200 micrograms/animal) was ineffective. There were no consistent patterns in plasma K. Renal clearance experiments showed that the same dose of aminoglutethimide decreased fractional tubular Na reabsorption and increased fractional tubular K reabsorption. Aldosterone reversed these trends suggesting that this hormone may play a role in controlling renal electrolyte excretion.