Plasma sex-steroid binding protein in a seasonally breeding reptile, Alligator mississippiensis

The properties of a sex-steroid binding protein (SSBP) in the plasma of the American alligator, Alligator mississippiensis, were partially characterized. Alligator SSBP has a sedimentation coefficient of 4S in a 5-20% sucrose gradient. It binds to estradiol-17 beta (E2) and testosterone (T) with limited capacities and moderate affinities (association constant for [3H]E2 is 4.70 +/- 0.09 X 10(8) M-1 and for [3H]T is 1.05 +/- 0.07 X 10(8) M-1, mean +/- SEM of six determinations). Plasma SSBP level, as measured by plasma [3H]E2 binding capacity, varies from 30 to 140 nmol per liter plasma (nM) and was found to be dependent on the gender, sexual maturity, and reproductive state of the animal. Distinct annual fluctuations in plasma SSBP level were observed in female alligators. In adult females, plasma SSBP levels were high (122 +/- 6 nM) in the fall during the nonbreeding season and low (30-60 nM) in spring and early summer during the breeding season. A minimum (33 +/- 6 nM) was reached in mid-June coinciding with the time of oviposition and rapid decline in circulating estrogen levels. This decline in adult female plasma SSBP levels during the breeding season was not observed in immature females. On the contrary, plasma SSBP levels in immature females increased from 81 +/- 14 nM in April to 134 +/- 9 nM in June. Plasma SSBP levels in male alligators showed little changes throughout the entire breeding season; they remained within the range of 80-100 nM from March to June. We believe that seasonal fluctuations in plasma SSBP levels constitute part of the mechanism involved in the regulation of free steroid delivered to target organs in female alligators and that such a mechanism does not exist in male animals.     

Testosterone-binding protein in reproductive tracts of fetal rats.

Testosterone-binding proteins may mediate the induction of Wolffian duct differentiation by testicular testosterone. The presence of such protein(s) was sought in reproductive tracts of 14.5-21.5-day-old fetal rats. Supernatant fractions (127,000 x g) were equilibrated with [3H]T) +/- radioinert testosterone in Tris - HCl:EDTA buffer, pH 7.4, (approximately 0.1 mg protein/0.5 ml) at 4 C for 16 hours. Bound and free (3H)T were separated by charcoaldextran adsorption or Sephadex G-100 gel filtration. The results with 14.5-15.0-day-old tracts were: a) specific binding to protein was saturated with increasing (3H)T concentration; b) Scatchard plot analysis indicated the presence of a single class of binding sites with high affinity (apparent Kd = 2 nM) and limited capacity (approximately 16 fmol/mg protein) for (3H)T; c) specific uptake was limited to (3H)T and (3H)5 alpha-dihydrotestosterone; d) (3H)T uptake by the tract supernatant was tissue-specific; e) pronase treatment abolished binding capacity for (3H)T; f) bound radioactivity consisted solely of (3H)T; and g) the mesonephric and ductal segment of the genital tract specifically binds (3H)T. The data demonstrate binding protein(s), specific for testosterone and possibly dihydrotestosterone, in the genital ducts of 14.5-15-day-old fetal rats. (3H)T binding to genital duct supernatants from male but not from female fetuses increased about 5-fold between 14.5 and 20.5 days of gestation. Upon Sephadex G-100 gel filtration, radioactivity was confined to the macromolecular fraction appearing in the void volume. Nuclear fractions, obtained from intact ducts incubated with (3H)T at 30 C but not at 0 C contained radioactivity. These observations are compatible with the existence of a cytoplasmic testosterone receptor or carrier protein aggregate. We have thus concluded that testosterone-binding proteins are present in the genital ducts of rat fetuses and that, in the male, their concentrations increase with progressive Wolffian duct differentiation.     

Alterations in spermatogenic activity and hormonal status in a seasonally breeding rat,Rattus fuscipes

The changes in the levels of serum FSH, LH, and testosterone have been studied during the seasonal reproductive cycle in males of the species Rattus fuscipes. In males captured in winter the seminiferous tubules were small, spermatogenesis was arrested at the primary spermatocyte stage, and the Sertoli cells contained increased numbers of lipid inclusions. The Leydig cells were atrophic and contained large crystalloids. The aspermatic state was accompanied by low levels of serum FSH, LH, and androgen. Reactivation of spermatogenesis occurred in spring and was accompanied by a rise in the levels of FSH, LH, and androgen. These hormonal changes were associated with a depletion of lipid inclusions from the Sertoli cells which paralleled the activation of spermatogenesis. The rising androgen levels were accompanied by the enlargement of Leydig cells and the disappearance of the crystalloids. In summer the fully active testes were associated with further increments of serum FSH and androgen levels above those seen during spring. It is concluded that the environmental cues controlling the seasonal reproductive cycle exert their influence on the testis through changes in gonadotrophin secretion by the pituitary gland.     

Leptin acts on metabolism in a photoperiod-dependent manner, but has no effect on reproductive function in the seasonally breeding Siberian hamster (Phodopus sungorus)

Leptin may play a role in appetite regulation and metabolism, but its reproductive role is less clear. In photoperiodic Siberian hamsters, seasonal changes in fatness, leptin gene expression, and metabolism occur synchronously with activation or suppression of reproduction, analogous to puberty. Here, we test the hypothesis that seasonal changes in leptin secretion mediate the photoperiodic regulation of reproduction. Mature male and ovariectomized estrogen-treated female Siberian hamsters were kept in long (LD; 16 h of light, 8 h of darkness) or short days (SD; 8 h of light, 16 h of darkness) for 8 weeks, and recombinant murine leptin (15 microg/day) was infused for 2 weeks via osmotic minipumps. SD hamsters exhibited significant weight and fat losses, reduced serum leptin and food intake, and suppressed pituitary LH concentration. Leptin did not suppress food intake over the 2-week treatment on either photoperiod, but significantly reduced fat reserves in SD hamsters. Leptin had no significant effect on pituitary LH concentrations in either sex or photoperiod or on testicular size and testosterone concentrations in males. These results suggest hamsters are more responsive to leptin on SD than on LD and that effects on food intake and fat loss can be dissociated in this species. Our data suggest that leptin does not mediate photoperiodic reproductive changes.     

The endogenous rhythm of plasma melatonin and its regulation by light in the highveld mole-rat (Cryptomys hottentotus pretoriae): a microphthalmic, seasonally breeding rodent

Abstract:  The day- and night-time levels of plasma melatonin were measured in adult male and female highveld mole-rats, Cryptomys hottentotus pretoriae . This study aimed to assess whether melatonin secretion in this nocturnal, strictly subterranean but seasonally breeding rodent has a day-night rhythm and whether that rhythm is circadian and can be modified by photoperiod. In experiment 1, a day-night rhythm of plasma melatonin was found in all animals housed on a 12L:12D schedule, with significantly higher concentrations in the dark (D) compared with the light (L) phase. The increment of plasma melatonin concentration at night was the same on days 1 and 2 for animals in the control group and animals transferred to constant dark. The animals transferred to constant light substantially reduced the amplitude of the melatonin rhythm on day 2. This suggests that the endogenous melatonin rhythm in C. h. pretoriae has a circadian pattern, which can be synchronized by photoperiod and inhibited by exposure to light at night. In experiment 2, the concentration of plasma melatonin in animals kept under 14L:10D (long day, LD) conditions differed significantly from animals on 10L:14D (short day, SD). This finding supports the notion that C. h. pretoriae is sensitive to changes in day length.     

Cable bacteria generate a firewall against euxinia in seasonally hypoxic basins

Seasonal oxygen depletion (hypoxia) in coastal bottom waters can lead to the release and persistence of free sulfide (euxinia), which is highly detrimental to marine life. Although coastal hypoxia is relatively common, reports of euxinia are less frequent, which suggests that certain environmental controls can delay the onset of euxinia. However, these controls and their prevalence are poorly understood. Here we present field observations from a seasonally hypoxic marine basin (Grevelingen, The Netherlands), which suggest that the activity of cable bacteria, a recently discovered group of sulfur-oxidizing microorganisms inducing long-distance electron transport, can delay the onset of euxinia in coastal waters. Our results reveal a remarkable seasonal succession of sulfur cycling pathways, which was observed over multiple years. Cable bacteria dominate the sediment geochemistry in winter, whereas, after the summer hypoxia, Beggiatoaceae mats colonize the sediment. The specific electrogenic metabolism of cable bacteria generates a large buffer of sedimentary iron oxides before the onset of summer hypoxia, which captures free sulfide in the surface sediment, thus likely preventing the development of bottom water euxinia. As cable bacteria are present in many seasonally hypoxic systems, this euxinia-preventing firewall mechanism could be widely active, and may explain why euxinia is relatively infrequently observed in the coastal ocean.The depletion of oxygen in bottom waters (hypoxia) is a naturally recurring phenomenon in some coastal systems (1), such as basins with restricted water circulation (2), and shelf regions subject to strong nutrient upwelling (3). Alongside this natural hypoxia, there is evidence for a global increase in the frequency, extent, intensity, and duration of coastal hypoxia, which is linked to an increased anthropogenic input of nutrients into the coastal ocean in combination with climate change (1, 4–6). The development of bottom water hypoxia has major consequences for the functioning of coastal ecosystems, sometimes leading to the formation of “dead zones” characterized by a complete absence of benthic fauna and fish. Areas sensitive to hypoxia are typically major fishing grounds, so the resulting economic and biodiversity losses make the global expansion of coastal hypoxia a subject of growing concern (7, 8).The ecosystem impacts of coastal hypoxia are particularly amplified when bottom water oxygen depletion progresses to a critical transition, termed euxinia, when free sulfide escapes from the sediment and accumulates in the bottom water (9). Even low levels of free sulfide are toxic to metazoan life, and therefore euxinia can induce mass mortality events, even among highly motile fauna like fish and large crustaceans (9–11). Although strong oxygen depletion, or even a complete removal of oxygen (anoxia), is often reported, concomitant reports of euxinia in coastal bottom waters are much scarcer. This suggests that certain sedimentary processes delay the onset of euxinia relative to anoxia, but, at present, the environmental controls on the timing and formation of coastal euxinia are poorly understood.Here we document a microbial mechanism that can delay or even prevent the development of euxinia in seasonally hypoxic basins. The mechanism is based on the metabolic activity of a newly discovered type of electrogenic microorganism, named cable bacteria (Desulfobulbaceae, Deltaproteobacteria), which are capable of inducing electrical currents over centimeter-scale distances in the sediment (12, 13). Cable bacteria have recently been suggested to be abundant in seasonally hypoxic coastal systems (14), but their impact on the biogeochemical cycling in these systems is unknown. These filamentous bacteria possess a unique respiratory metabolism in which the oxidative removal of sulfide in deeper sediment layers is electrically coupled to the reductive consumption of oxygen just below the sediment–water interface (12), a process referred to as electrogenic sulfur oxidation (e-SOx) (15). In laboratory experiments, e-SOx has been shown to exert a strong impact on sedimentary iron and sulfur cycling, leading to a conversion of iron sulfides into iron oxides (16). Here we demonstrate that the same interconversion process of iron minerals occurs in the sediments of a seasonally hypoxic marine basin, and that the large pool of iron oxides can act as a “firewall,” which can substantially delay the development of euxinia.     

Evidence for a proenkephalin-like precursor in amphibian brain.

The mammalian proenkephalin-derived peptides [Leu]enkephalin, [Met]enkephalin, and [Met]enkephalin-Arg6-Phe7 were identified in acid extracts of the brain of Bufo marinus by using reversed-phase HPLC and specific radioimmunoassays. [Met]Enkephalin was the predominant opioid peptide present (270 pmol/g). In contrast, the octapeptide [Met]enkephalin-Arg6-Gly7-Leu8, which is also derived from mammalian proenkephalin, was not detected. The ratio of free [Met]enkephalin to [Met]enkephalin-Arg6-Phe7 was found to be 3.5 to 1, which is similar to that observed in mammalian proenkephalin-containing tissues. Together these data (i) indicate that amphibian brain contains a proenkephalin related to the mammalian precursor and (ii) establish the existence of enkephalins and proenkephalin-derived enkephalin-containing peptides in a submammalian species.     

Cholecystokinin activation of central satiety centers changes seasonally in a mammalian hibernator

Hibernators that rely on lipids during winter exhibit profound changes in food intake over the annual cycle. The mechanisms that regulate appetite changes in seasonal hibernators remain unclear, but likely consist of complex interactions between gut hormones, adipokines, and central processing centers. We hypothesized that seasonal changes in the sensitivity of neurons in the nucleus tractus solitarius (NTS) to the gut hormone cholecystokinin (CCK) may contribute to appetite regulation in ground squirrels. Spring (SPR), late summer (SUM), and winter euthermic hibernating (HIB) 13-lined ground squirrels (Ictidomys tridecemlineatus) were treated with intraperitoneal CCK (100 μg/kg) or vehicle (CON) for 3 h and Fos expression in the NTS was quantified. In CON squirrels, numbers of Fos-positive neurons in HIB were low compared to SPR and SUM. CCK treatment increased Fos-positive neurons in the NTS at the levels of the area postrema (AP) and pre AP during all seasons and at the level of the rostral AP in HIB squirrels. The highest absolute levels of Fos-positive neurons were found in SPR CCK squirrels, but the highest relative increase from CON was found in HIB CCK squirrels. Fold-changes in Fos-positive neurons in SUM were intermediate between SPR and HIB. Thus, CCK sensitivity falls from SPR to SUM suggesting that seasonal changes in sensitivity of NTS neurons to vagally-derived CCK may influence appetite in the active phase of the annual cycle in hibernating squirrels. Enhanced sensitivity to CCK signaling in NTS neurons of hibernators indicates that changes in gut-brain signaling may contribute to seasonal changes in food intake during the annual cycle.     

Cultured cells as a model for amphibian metamorphosis.

Gene expression screens have been applied to a cultured cell line of Xenopus laevis, XL-177, to isolate genes that are up- and down-regulated in the first 8 h after thyroid hormone (TH) induction. At least 14 up-regulated genes were isolated from TH-induced cells grown in the presence or absence of cycloheximide, an inhibitor of protein synthesis. These genes respond directly to TH as demonstrated by the resistance of up-regulation to protein synthesis inhibition in the cultured cells or in tadpoles. Kinetics of mRNA accumulation after TH induction is similar for these genes, including those that are superinduced by cycloheximide. Their mRNAs start to be up-regulated several hours after TH treatment and reach maximum levels between 8 and 16 h. These genes show up-regulation in one or more tadpole organs in response to exogenous TH. Only a few minimally down-regulated genes were identified. Fourteen of the 20 genes that were found to be up-regulated by TH in tadpole tail are also up-regulated in XL-177 cells. Their up-regulation falls into the same two kinetic patterns in the cultured cells as it does in tadpole tail. Another cell line of X. laevis, XLA, is greatly reduced in its ability to up-regulate the same genes isolated from XL-177 cells and tadpole tails in response to TH. Thus these cell lines make up a model system to examine the interactions of gene expression triggered by TH during amphibian metamorphosis.     

Luteinizing hormone-releasing hormone involvement in the reproductive behavior of a male amphibian

The present study demonstrates that an intracerebroventricular injection of luteinizing hormone-releasing hormone (LHRH) activates sexual behaviors and elevates the plasma androgen concentrations in rough-skinned newts (Taricha granulosa). The stimulatory effect of LHRH on male behavior may be a seasonal phenomenon, because LHRH stimulation of sexual behavior was only observed during the early part of the breeding season (November and December). When an active LHRH antagonist was injected into male newts, sexual behavior was suppressed. These studies indicate that endogenous LHRH is involved in regulating sexual behavior in this amphibian.     

Receptors for native gonadotropins in amphibian liver

We demonstrate that highly purified bullfrog (f) follicle-stimulating hormone (FSH) and luteinizing hormone (LH) bind specifically and significantly to a crude plasma membrane fraction of bullfrog liver. The other extragonadal organs of the bullfrog showed little or no specific binding. Specific bindings of 125I-fFSH and 125I-fLH to plasma membranes are saturable processes, and are time-, pH-, and temperature-dependent. Scatchard plots of fFSH and fLH were linear. The association constant of equilibrium (Ka) of the specific fFSH binding sites was 4.77 +/- 1.24 X 10(9)M-1 (mean +/- SEM) and the number of sites was 0.262 +/- 0.042 fmol/mg protein (mean +/- SEM). The Ka of the specific fLH binding sites was 5.38 +/- 1.27 X 10(9)M-1 (mean +/- SEM) and the number was 0.315 +/- 0.019 fmol/mg protein (mean +/- SEM). Competition experiments revealed that both fFSH and fLH use the same single class of binding sites. Binding of rat, chicken, bullfrog, and salmon gonadotropins to plasma membranes of the testis and liver of various vertebrates was studied. A significant degree of specific binding was detected only in combinations of bullfrog gonadotropins and amphibian livers. The concentration of adenosine 3'-5'-monophosphate (cAMP) in mince or primary culture cells of bullfrog liver was greatly increased by adding fFSH and fLH to the medium. Bullfrog LH was more potent than fFSH in increasing cAMP concentration, although they were not distinguished by specific binding sites. These data suggest that not only the gonads but also the liver is the target of gonadotropins in the bullfrog, although the final hepatic function controlled by gonadotropins remains unknown.     

Emerging infectious disease and the loss of biodiversity in a Neotropical amphibian community

Pathogens rarely cause extinctions of host species, and there are few examples of a pathogen changing species richness and diversity of an ecological community by causing local extinctions across a wide range of species. We report the link between the rapid appearance of a pathogenic chytrid fungus Batrachochytrium dendrobatidis in an amphibian community at El Copé, Panama, and subsequent mass mortality and loss of amphibian biodiversity across eight families of frogs and salamanders. We describe an outbreak of chytridiomycosis in Panama and argue that this infectious disease has played an important role in amphibian population declines. The high virulence and large number of potential hosts of this emerging infectious disease threaten global amphibian diversity.     

Fish melanin-concentrating hormone disperses melanin in amphibian melanophores

Melanin-concentrating hormone (MCH), which was isolated from salmon pituitary and caused melanin concentration in fish scale melanophores, has been tested on cultured chromatophores of an amphibian, the bullfrog tadpole. MCH induced dispersion of melanin in cultured melanophores of the tadpole. The duration of the dispersing effect of MCH was relatively short compared with that of alpha-melanocyte-stimulating hormone (alpha-MSH). MCH also induced the concentration of cultured iridophores of bullfrog tadpole.     

Synergism between UV-B radiation and a pathogen magnifies amphibian embryo mortality in nature.

Previous research has shown that amphibians have differential sensitivity to ultraviolet-B (UV-B) radiation. In some species, ambient levels of UV-B radiation cause embryonic mortality in nature. The detrimental effects of UV-B alone or with other agents may ultimately affect amphibians at the population level. Here, we experimentally demonstrate a synergistic effect between UV-B radiation and a pathogenic fungus in the field that increases the mortality of amphibian embryos compared with either factor alone. Studies investigating single factors for causes of amphibian egg mortality or population declines may not reveal the complex factors involved in declines.     

Multiple forms of gonadotropin-releasing hormone in amphibian brains

Several forms of gonadotropin-releasing hormone (GnRH)-like molecules were found in brains of both anurans (frogs) and urodeles (salamanders). The presence of the mammalian-like GnRH molecule was confirmed by HPLC and cross-reactivity studies. Small amounts of salmonid-like GnRH molecules in the brains of frogs (Rana pipiens, Hyla regilla) and salamanders (Taricha granulosa, Ambystoma gracile) were detected by comparing the HPLC chromatographic pattern and immunological reactivity of the brain extracts with native trout and synthetic salmon GnRH. This nonmammalian form of GnRH in the amphibian brain is similar and perhaps identical, at least by indirect evidence, to a form of GnRH reported earlier to be in sympathetic ganglion, retina, chromaffin tissue, and tadpole brain. If two of the amphibian GnRH molecules prove to be mammalian and salmon GnRH, then it is likely that two separate genes in amphibians code for the distinct primary structures of the molecules. The most parsimonious interpretation of the presence of both mammalian- and salmon-like GnRH in anurans and urodeles is that a common phylogenetic ancestor also possessed the two forms of GnRH. Thus the mammalian form of GnRH may well have been present in labyrinthodont amphibians. Independent of evolutionary origin, the functions of the different GnRH molecules in amphibians are unknown.