Short-term exposure to high pCO2 leads to decreased branchial cytochrome C oxidase activity in the presence of octopamine in a decapod.

In a recent mechanistic study, octopamine was shown to promote proton transport over the branchial epithelium in green crabs, Carcinus maenas. Here, we follow up on this finding by investigating the involvement of octopamine in an environmental and physiological context that challenges acid-base homeostasis, the response to short-term high pCO2 exposure (400 Pa) in a brackish water environment. We show that hyperregulating green crabs experienced a respiratory acidosis as early as 6 h of exposure to hypercapnia, with a rise in hemolymph pCO2 accompanied by a simultaneous drop of hemolymph pH. The slightly delayed increase in hemolymph HCO3- observed after 24 h helped to restore hemolymph pH to initial values by 48 h. Circulating levels of the biogenic amine octopamine were significantly higher in short-term high pCO2 exposed crabs compared to control crabs after 48 h. Whole animal metabolic rates, intracellular levels of octopamine and cAMP, as well as branchial mitochondrial enzyme activities for complex I + III and citrate synthase were unchanged in posterior gill #7 after 48 h of hypercapnia. However, application of octopamine in gill respirometry experiments suppressed branchial metabolic rate in posterior gills of short-term high pCO2 exposed animals. Furthermore, branchial enzyme activity of cytochrome C oxidase decreased in high pCO2 exposed crabs after 48 h. Our results indicate that hyperregulating green crabs are capable of quickly counteracting a hypercapnia-induced respiratory acidosis. The role of octopamine in the acclimation of green crabs to short-term hypercapnia seems to entail the alteration of branchial metabolic pathways, possibly targeting mitochondrial cytochrome C in the gill. Our findings help advancing our current limited understanding of endocrine components in hypercapnia acclimation. SUMMARY STATEMENT: Acid-base compensation upon short-term high pCO2 exposure in hyperregulating green crabs started after 6 h and was accomplished by 48 h with the involvement of the biogenic amine octopamine, accumulation of hemolymph HCO3-, and regulation of mitochondrial complex IV (cytochrome C oxidase).

Branchial CO2 and ammonia excretion in crustaceans: Involvement of an apical Rhesus-like glycoprotein.

AIM: To determine whether the crustacean Rh1 protein functions as a dual CO2 /ammonia transporter and investigate its role in branchial ammonia excretion and acid-base regulation. METHODS: Sequence analysis of decapod Rh1 proteins was used to determine the conservation of amino acid residues putatively involved in ammonia transport and CO2 binding in human and bacterial Rh proteins. Using the Carcinus maenas Rh1 protein (CmRh1) as a representative of decapod Rh1 proteins, we test the ammonia and CO2 transport capabilities of CmRh1 through heterologous expression in yeast and Xenopus oocytes coupled with site-directed mutagenesis. Quantitative PCR was used to assess the distribution of CmRh1 mRNA in various tissues. Western blotting was used to assess CmRh1 protein expression changes in response to high environmental ammonia and CO2 . Further, immunohistochemistry was used to assess sub-cellular localization of CmRh1 and a membrane-bound carbonic anhydrase (CmCAg). RESULTS: Sequence analysis of decapod Rh proteins revealed high conservation of several amino acid residues putatively involved in conducting ammonia transport and CO2 binding. Expression of CmRh1 in Xenopus oocytes enhanced both ammonia and CO2 transport which was nullified in CmRh1 D180N mutant oocytes. Transport of the ammonia analog methylamine by CmRh1 is dependent on both ionized and un-ionized ammonia/methylamine species. CmRh1 was co-localized with CmCAg to the apical membrane of the crustacean gill and only experienced decreased protein expression in the anterior gills when exposed to high environmental ammonia. CONCLUSION: CmRh1 is the first identified apical transporter-mediated route for ammonia and CO2 excretion in the crustacean gill. Our findings shed further light on the potential universality of dual ammonia and CO2 transport capacity of Rhesus glycoproteins in both vertebrates and invertebrates.

The role of octopamine and crustacean hyperglycemic hormone (CHH) in branchial acid-base regulation in the European green crab, Carcinus maenas.

Crustaceans' endocrinology is a vastly understudied area of research. The major focus of the studies on this topic to date has been on the molting cycle (and in particular, the role of crustacean hyperglycemic hormone (CHH)), as well as the role of other hormones in facilitating physiological phenotypic adjustments to salinity changes. Additionally, while many recent studies have been conducted on the acclimation and adaptation capacity of crustaceans to a changing environment, only few have investigated internal hormonal balance especially with respect to an endocrine response to environmental challenges. Consequently, our study aimed to identify and characterize endocrine components of acid-base regulation in the European green crab, Carcinus maenas. We show that both the biogenic amine octopamine (OCT) and the CHH are regulatory components of branchial acid-base regulation. While OCT suppressed branchial proton excretion, CHH seemed to promote it. Both hormones were also capable of enhancing branchial ammonia excretion. Furthermore, mRNA abundance for branchial receptors (OCT-R), or G-protein receptor activated soluble guanylate cyclase (sGC1b), are affected by environmental change such as elevated pCO2 (hypercapnia) and high environmental ammonia (HEA). Our findings support a role for both OCT and CHH in the general maintenance of steady-state acid-base maintenance in the gill, as well as regulating the acid-base response to environmental challenges that C. maenas encounters on a regular basis in the habitats it dwells in and more so in the future ocean.

Hiat1 as a new transporter involved in ammonia regulation.

The orphan transporter hippocampus-abundant transcript 1 (Hiat1) was first identified in the mammalian brain. Its specific substrate specificity, however, has not been investigated to date. Here, we identified and analyzed Hiat1 in a crustacean, the green crab Carcinus maenas. Our phylogenetic analysis showed that Hiat1 protein is conserved at a considerable level between mammals and this invertebrate (ca. 78% identical and conserved amino acids). Functional expression of Carcinus maenas Hiat1 in Xenopus laevis oocytes demonstrated the capability to transport ammonia (likely NH4+) in a sodium-dependent manner. Furthermore, applying quantitative polymerase chain reaction, our results indicated a physiological role for Carcinus maenas Hiat1 in ammonia homeostasis, as mRNA abundance increased in posterior gills in response to elevated circulating hemolymph ammonia upon exposure to high environmental ammonia. Its ubiquitous mRNA expression pattern also suggests an essential role in general cellular detoxification of ammonia. Overall, our results introduce a new ubiquitously expressed ammonia transporter, consequently demanding revision of our understanding of ammonia handling in key model systems from mammalian kidneys to crustacean and fish gills.

Characterization of two novel ammonia transporters, Hiat1a and Hiat1b, in the teleost model system Danio rerio.

Ammonia excretion in fish excretory epithelia is a complex interplay of multiple membrane transport proteins and mechanisms. Using the model system of zebrafish (Danio rerio) larvae, here we identified three paralogues of a novel ammonia transporter, hippocampus-abundant transcript 1 (DrHiat1), also found in most vertebrates. When functionally expressed in Xenopus laevis oocytes, DrHiat1a and DrHiat1b promoted methylamine uptake in a competitive manner with ammonia. In situ hybridization experiments showed that both transporters were expressed as early as the 4-cell stage in zebrafish embryos and could be identified in most tissues 4 days post-fertilization. Larvae experiencing morpholino-mediated knockdown of DrHiat1b exhibited significantly lower whole-body ammonia excretion rates compared with control larvae. Markedly decreased site-specific total ammonia excretion of up to 85% was observed in both the pharyngeal region (site of developing gills) and the yolk sac (region shown to have the highest NH4+ flux). This study is the first to identify DrHiat1b/DrHIAT1 in particular as an important contributor to ammonia excretion in larval zebrafish. Being evolutionarily conserved, these proteins are likely involved in multiple other general ammonia-handling mechanisms, making them worthy candidates for future studies on nitrogen regulation in fishes and across the animal kingdom.

A potential role for hyperpolarization-activated cyclic nucleotide-gated sodium/potassium channels (HCNs) in teleost acid-base and ammonia regulation.

Increasing evidence suggests the involvement of hyperpolarization-activated cyclic nucleotide-gated sodium/potassium channels (HCNs) not only in cardiac and neural function, but also in more general physiological processes including acid-base and ammonia regulation. We have identified four different HCN paralogs/isoforms in the goldfish Carassius auratus (CaHCN1, CaHCN2b, CaHCN4a and CaHCN4b) as likely candidates to contribute to renal, branchial and intestinal acid-base and ammonia regulation in this teleost. Quantitative real-time PCR showed not only high mRNA abundance of all isoforms in heart and brain, but also detectable levels (particularly of CaHCN2b and CaHCN4b) in non-excitable tissues, including gills and kidneys. In response to an internal or external acid-base and/or ammonia disturbance caused by feeding or high environmental ammonia, respectively, we observed differential and tissue-specific changes in mRNA abundance of all isoforms except CaHCN4b. Furthermore, our data suggest that the functions of specific HCN channels are supplemented by certain Rhesus glycoprotein functions to help in the protection of tissues from elevated ammonia levels, or as potential direct routes for ammonia transport in gills, kidney, and gut. The present results indicate important individual roles for each HCN isoform in response to acid-base and ammonia disturbances.

Is ammonia excretion affected by gill ventilation in the rainbow trout Oncorhynchus mykiss?

Ammonia (NH3 + NH4+) is the major nitrogenous waste in teleost fish. NH3 is also the third respiratory gas, playing a role in ventilatory control. However it is also highly toxic. Normally, ammonia excretion through the gills occurs at about the same rate as its metabolic production, but the branchial transport mechanisms have long been controversial. An influential review in this journal has claimed that ammonia excretion in fish is probably limited by diffusion rather than by convection, so that increases in ventilation would have negligible effect on the rate of ammonia excretion. Why then should elevated plasma ammonia stimulate ventilation? The diffusion-limitation argument was made before the discovery of Rhesus (Rh) glycoproteins and the associated metabolon in the gills, which serve to greatly increase branchial ammonia permeability under conditions of ammonia loading. Therefore, we hypothesized here that (i) in accord with the diffusion-limitation concept, changes in ventilation would not affect the rate of ammonia excretion under conditions where branchial Rh metabolon function would be low (resting trout with low plasma ammonia levels). However, we also hypothesized that (ii) in accord with convective limitation, changes in ventilation would influence the rate of ammonia excretion under conditions where diffusion limitation was removed because branchial Rh metabolon function would be high (ammonia-loaded trout with high plasma ammonia levels). We used variations in environmental O2 levels to manipulate ventilation in trout under control or ammonia-loaded conditions - i.e. hyperventilation in moderate hypoxia or hypoventilation in moderate hyperoxia. In accord with hypothesis (i), under resting conditions, ammonia excretion was insensitive to experimentally induced changes in ventilation. Ammonia-loading by NH4HCO3 infusion for 30h + increased the gill mRNA expressions of two key metabolon components (Rhcg2, V-H+-ATPase or HAT), together with a 7.5-fold increase in plasma ammonia concentration and a 3-fold increase in ammonia excretion rate. In accord with hypothesis (ii), in these fish, hypoxia-induced increases in ventilation elevated the ammonia excretion rate and lowered plasma ammonia, while hyperoxia-induced decreases in ventilation reduced the ammonia excretion rate, and elevated plasma ammonia concentration. We conclude that under conditions of natural ammonia loading (e.g. meal digestion, post-exercise recovery), diffusion-limitation is removed by Rh metabolon upregulation, such that the stimulation of ventilation by elevated plasma ammonia can play an important role in clearing the potentially toxic ammonia load.

Section-specific H+ flux in renal tubules of fasted and fed goldfish.

A recent study demonstrated that in response to a feeding-induced metabolic acidosis, goldfish (Carassius auratus) adjust epithelial protein and/or mRNA expression in their kidney tubules for multiple transporters known to be relevant for acid-base regulation. These include Na+/H+ exchanger (NHE), V-type H+-ATPase (V-ATPase), cytoplasmic carbonic anhydrase, HCO3- transporters and Rhesus proteins. Consequently, renal acid output in the form of protons and NH4+ increases. However, little is known about the mechanistic details of renal acid-base regulation in C. auratus and teleost fishes in general. The present study applied the scanning ion-selective electrode technique (SIET) to measure proton flux in proximal, distal and connecting tubules of goldfish. We detected increased H+ efflux into the extracellular fluid from the tubule in fed animals, resulting from paracellular back-flux of H+ through the tight junction. By applying inhibitors for selected acid-base regulatory epithelial transporters, we found that cytosolic carbonic anhydrase and HCO3- transporters were important in mediating H+ flux in all three tubule segments of fed goldfish. Contrastingly, V-ATPase seemed to play a role in H+ flux only in proximal and distal tubules, and NHE in proximal and connecting tubules. We developed working models for transport of acid-base relevant equivalents (H+, HCO3-, NH3/NH4+) for each tubule segment in C. auratus kidney. While the proximal tubule appears to play a major role in both H+ secretion and HCO3- reabsorption, the distal and connecting tubules seem to mainly serve for HCO3- reabsorption and NH3/NH4+ secretion.

Section-specific expression of acid-base and ammonia transporters in the kidney tubules of the goldfish Carassius auratus and their responses to feeding.

In teleost fishes, renal contributions to acid-base and ammonia regulation are often neglected compared with the gills. In goldfish, increased renal acid excretion in response to feeding was indicated by increased urine ammonia and inorganic phosphate concentrations and decreased urine pH. By microdissecting the kidney tubules and performing quantitative real-time PCR and/or immunohistochemistry, we profiled the section-specific expression of glutamate dehydrogenase (GDH), glutamine synthetase (GS), Na+/H+-exchanger 3 (NHE3), carbonic anhydrase II (CAIIa), V-H+-ATPase subunit 1b, Cl-/ HCO3- -exchanger 1 (AE1), Na+/ HCO3- -cotransporter 1 (NBC1), Na+/K+-ATPase subunit 1α, and Rhesus-proteins Rhbg, Rhcg1a, and Rhcg1b. Here, we show for the first time that 1) the proximal tubule appears to be the major site for ammoniagenesis, 2) epithelial transporters are differentially expressed along the renal tubule, and 3) a potential feeding-related "acidic tide" results in the differential regulation of epithelial transporters, resembling the mammalian renal response to a metabolic acidosis. Specifically, GDH and NHE3 mRNAs were upregulated and GS downregulated in the proximal tubule upon feeding, suggesting this section as a major site for ammoniagenesis and acid secretion. The distal tubule may play a major role in renal ammonia secretion, with feeding-induced upregulation of mRNA and protein for apical NHE3, cytoplasmic CAIIa, universal Rhcg1a and apical Rhcg1b, and downregulation of basolateral Rhbg and AE1. Changes in mRNA expression of the Wolffian ducts and bladder suggest supporting roles in fine-tuning urine composition. The present study verifies an important renal contribution to acid-base balance and emphasizes that studies looking at the whole kidney may overlook key section-specific responses.

Effects of salinity on short-term waterborne zinc uptake, accumulation and sub-lethal toxicity in the green shore crab (Carcinus maenas).

Waterborne zinc (Zn) is known to cause toxicity to freshwater animals primarily by disrupting calcium (Ca) homeostasis during acute exposure, but its effects in marine and estuarine animals are not well characterized. The present study investigated the effects of salinity on short-term Zn accumulation and sub-lethal toxicity in the euryhaline green shore crab, Carcinus maenas. The kinetic and pharmacological properties of short-term branchial Zn uptake were also examined. Green crabs (n=10) were exposed to control (no added Zn) and 50µM (3.25mgL(-1)) of waterborne Zn (∼25% of 96h LC50 in 100 seawater) for 96h at 3 different salinity regimes (100%, 60% and 20% seawater). Exposure to waterborne Zn increased tissue-specific Zn accumulation across different salinities. However, the maximum accumulation occurred in 20% seawater and no difference was recorded between 60% and 100% seawater. Gills appeared to be the primary site of Zn accumulation, since the accumulation was significantly higher in the gills relative to the hepatopancreas, haemolymph and muscle. Waterborne Zn exposure induced a slight increase in haemolymph osmolality and chloride levels irrespective of salinity. In contrast, Zn exposure elicited marked increases in both haemolymph and gill Ca levels, and these changes were more pronounced in 20% seawater relative to that in 60% or 100% seawater. An in vitro gill perfusion technique was used to examine the characteristics of short-term (1-4h) branchial Zn uptake over an exposure concentration range of 3-12µM (200-800µgL(-1)). The rate of short-term branchial Zn uptake did not change significantly after 2h, and no difference was recorded in the rate of uptake between the anterior (respiratory) and posterior (ion transporting) gills. The in vitro branchial Zn uptake occurred in a concentration-dependent manner across different salinities. However, the rate of uptake was consistently higher in 20% seawater relative to 60% or 100% seawater - similar to the trend observed with tissue Zn accumulation during in vivo exposure. The short-term branchial Zn uptake was found to be inhibited by lanthanum (a blocker of voltage-independent Ca channels), suggesting that branchial Zn uptake occurs via the Ca transporting pathways, at least in part. Overall, our findings indicate that acute exposure to waterborne Zn leads to the disruption of Zn and Ca homeostasis in green crab, and these effects are exacerbated at the lower salinity.

The role of an ancestral hyperpolarization-activated cyclic nucleotide-gated K+ channel in branchial acid-base regulation in the green crab, Carcinus maenas.

Numerous electrophysiological studies on branchial K(+) transport in brachyuran crabs have established an important role for potassium channels in osmoregulatory ion uptake and ammonia excretion in the gill epithelium of decapod crustaceans. However, hardly anything is known of the actual nature of these channels in crustaceans. In the present study, the identification of a hyperpolarization-activated cyclic nucleotide-gated potassium channel (HCN) in the transcriptome of the green crab Carcinus maenas and subsequent performance of quantitative real-time PCR revealed the ubiquitous expression of this channel in this species. Even though mRNA expression levels in the cerebral ganglion were found to be approximately 10 times higher compared with all other tissues, posterior gills still expressed significant levels of HCN, indicating an important role for this transporter in branchial ion regulation. The relatively unspecific K(+)-channel inhibitor Ba(2+), as well as the HCN-specific blocker ZD7288, as applied in gill perfusion experiments and electrophysiological studies employing the split gill lamellae revealed the presence of at least two different K(+)/NH4(+)-transporting structures in the branchial epithelium of C. maenas. Furthermore, HCN mRNA levels in posterior gill 7 decreased significantly in response to the respiratory or metabolic acidosis that was induced by acclimation of green crabs to high environmental PCO2 and ammonia, respectively. Consequently, the present study provides first evidence that HCN-promoted NH4(+) epithelial transport is involved in both branchial acid-base and ammonia regulation in an invertebrate.

Making sense of nickel accumulation and sub-lethal toxic effects in saline waters: Fate and effects of nickel in the green crab, Carcinus maenas.

In freshwater, invertebrates nickel (Ni) is considered an ionoregulatory toxicant, but its mechanism of toxicity in marine settings, and how this varies with salinity, is poorly understood. This study investigated Ni accumulation and physiological mechanisms of sub-lethal Ni toxicity in the euryhaline green crab Carcinus maenas. Male crabs were exposed to 8.2µg/L (the US EPA chronic criterion concentration for salt waters) of waterborne Ni (radiolabelled with (63)Ni) at three different salinities, 20%, 60% and 100% SW for 24h. Whole body Ni accumulation in 20% SW was 3-5 fold greater than in 60% or 100% SW, and >80% of accumulated Ni was in the carapace at all salinities. Ni also accumulated in posterior gill 8, which showed a higher accumulation in 20% SW than in other salinities, a pattern also seen at higher exposure concentrations of Ni (500 and 3000µg/L). Gill perfusion experiments revealed that Ni was taken up by both anterior and posterior gills, but in 20% SW the posterior gill 8, which performs ionoregulatory functions, accumulated more Ni than the anterior gill 5, which primarily has a respiratory function. The sub-lethal consequences of Ni exposure were investigated by placing crabs in Ni concentrations of 8.2, 500, and 3000µg/L at 20, 60 or 100% SW for 24h. In 20% SW, haemolymph Ca levels were significantly decreased by exposure to Ni concentrations of 8.2µg/L or higher, whereas Na concentrations were depressed only at 3000µg/L. Na(+)/K(+)-ATPase activity was inhibited at both 500 and 3000µg/L in gill 8, but only in 20% SW. Haemolymph K, Mg, and osmolality were unaffected throughout, though all varied with salinity in the expected fashion. These data suggest that Ni impacts ionoregulatory function in the green crab, in a gill- and salinity-dependent manner.

Cutaneous nitrogen excretion in the African clawed frog Xenopus laevis: effects of high environmental ammonia (HEA).

Ammonia is a highly toxic molecule and often introduced in considerable amounts into aquatic environments due to anthropogenic activities. Many aquatic and semi-aquatic amphibians utilize, in addition to their kidneys, the skin for osmoregulation and nitrogen excretion. In the present study the effects of prolonged (7-21 days) exposure to high environmental ammonia (HEA, 1 mmol l(-1) NH4Cl) on cutaneous nitrogen excretion and gene expression of key-transporters involved in nitrogen excretion and acid-base regulation were investigated in the fully aquatic African clawed frog, Xenopus laevis. The study revealed that X. laevis excretes predominately ammonia of which approximately 50% is excreted via the skin. Both the ventral and dorsal skin were capable to generate a net ammonia efflux, which was significantly activated by 10 mmol l(-1) of the phosphodiesterase blocker theophylline. The obtained data further suggest that the ammonia efflux was promoted by an acidification of the unstirred boundary layer, likely generated by an apical localized V-ATPase, with NH3 being transported via cutaneous expressed ammonia transporters, Rhbg and Rhcg. Prolonged HEA exposure did significantly reduce the net-flux rates over the ventral skin with Vmax changing from 256 nmol cm(-2) h(-1) in control frogs to 196 nmol cm(-2) h(-1) in HEA exposed animals. Further, prolonged HEA exposure caused a decrease in mRNA expression levels of the ammonia transporter Rhbg, Na(+)/K(+)-ATPase (α-subunit) and V-ATPase (subunit H) in the ventral and dorsal skin and the kidney. In contrast, Rhcg expression levels were unaffected by HEA in skin tissues.

Differential acid-base regulation in various gills of the green crab Carcinus maenas: Effects of elevated environmental pCO2.

Euryhaline decapod crustaceans possess an efficient regulation apparatus located in the gill epithelia, providing a high adaptation potential to varying environmental abiotic conditions. Even though many studies focussed on the osmoregulatory capacity of the gills, acid-base regulatory mechanisms have obtained much less attention. In the present study, underlying principles and effects of elevated pCO(2) on acid-base regulatory patterns were investigated in the green crab Carcinus maenas acclimated to diluted seawater. In gill perfusion experiments, all investigated gills 4-9 were observed to up-regulate the pH of the hemolymph by 0.1-0.2 units. Anterior gills, especially gill 4, were identified to be most efficient in the equivalent proton excretion rate. Ammonia excretion rates mirrored this pattern among gills, indicating a linkage between both processes. In specimen exposed to elevated pCO(2) levels for at least 7 days, mimicking a future ocean scenario as predicted until the year 2300, hemolymph K(+) and ammonia concentrations were significantly elevated, and an increased ammonia excretion rate was observed. A detailed quantitative gene expression analysis revealed that upon elevated pCO(2) exposure, mRNA levels of transcripts hypothesized to be involved in ammonia and acid-base regulation (Rhesus-like protein, membrane-bound carbonic anhydrase, Na(+)/K(+)-ATPase) were affected predominantly in the non-osmoregulating anterior gills.

Effects of elevated seawater pCO(2) on gene expression patterns in the gills of the green crab, Carcinus maenas.

BACKGROUND: The green crab Carcinus maenas is known for its high acclimation potential to varying environmental abiotic conditions. A high ability for ion and acid-base regulation is mainly based on an efficient regulation apparatus located in gill epithelia. However, at present it is neither known which ion transport proteins play a key role in the acid-base compensation response nor how gill epithelia respond to elevated seawater pCO(2) as predicted for the future. In order to promote our understanding of the responses of green crab acid-base regulatory epithelia to high pCO(2), Baltic Sea green crabs were exposed to a pCO(2) of 400 Pa. Gills were screened for differentially expressed gene transcripts using a 4,462-feature microarray and quantitative real-time PCR. RESULTS: Crabs responded mainly through fine scale adjustment of gene expression to elevated pCO(2). However, 2% of all investigated transcripts were significantly regulated 1.3 to 2.2-fold upon one-week exposure to CO(2) stress. Most of the genes known to code for proteins involved in osmo- and acid-base regulation, as well as cellular stress response, were were not impacted by elevated pCO(2). However, after one week of exposure, significant changes were detected in a calcium-activated chloride channel, a hyperpolarization activated nucleotide-gated potassium channel, a tetraspanin, and an integrin. Furthermore, a putative syntaxin-binding protein, a protein of the transmembrane 9 superfamily, and a Cl(-)/HCO(3)(-) exchanger of the SLC 4 family were differentially regulated. These genes were also affected in a previously published hypoosmotic acclimation response study. CONCLUSIONS: The moderate, but specific response of C. maenas gill gene expression indicates that (1) seawater acidification does not act as a strong stressor on the cellular level in gill epithelia; (2) the response to hypercapnia is to some degree comparable to a hypoosmotic acclimation response; (3) the specialization of each of the posterior gill arches might go beyond what has been demonstrated up to date; and (4) a re-configuration of gill epithelia might occur in response to hypercapnia.

Effects of high environmental ammonia on branchial ammonia excretion rates and tissue Rh-protein mRNA expression levels in seawater acclimated Dungeness crab Metacarcinus magister.

In the present study of the marine Dungeness crabs Metacarcinus magister, the long term effects of high environmental ammonia (HEA) on hemolymph ammonia and urea concentrations, branchial ammonia excretion rates and mRNA expression levels of the crustacean Rh-like ammonia transporter (RhMM), H(+)-ATPase (subunit B), Na(+)/K(+)-ATPase (α-subunit) and Na(+)/H(+)-exchanger (NHE) were investigated. Under control conditions, the crabs' hemolymph exhibited a total ammonia concentration of 179.3±14.5µmol L(-1), while urea accounted for 467.2±33.5µmol L(-1), respectively. Both anterior and posterior gills were capable of excreting ammonia against a 16-fold inwardly directed gradient. Under control conditions, mRNA expression levels of RhMM were high in the gills in contrast to very low expression levels in all other tissues investigated, including the antennal gland, hepatopancreas, and skeletal muscle. After exposure to 1mmol L(-1) NH(4)Cl, hemolymph ammonia increased within the first 12h to ca. 500µmol L(-1) and crabs were able the keep this hemolymph ammonia level for at least 4 days. During this initial period, branchial RhMM and H(+)-ATPase (subunit B) mRNA expression levels roughly doubled. After 14 days of HEA exposure, hemolymph ammonia raised up to environmental levels, whereas urea levels increased by ca. 30%. At the same time, whole animal ammonia and urea excretion vanished. Additionally, branchial RhMM, H(+)-ATPase, Na(+)/K(+)-ATPase and NHE mRNA levels decreased significantly after long term HEA exposure, whereas expression levels of RhMM in the internal tissues increased substantially. Interestingly, crabs acclimated to HEA showed no mortality even after 4 weeks of HEA exposure. This suggests that M. magister possesses a highly adaptive mechanism to cope with elevated ammonia concentrations in its body fluids, including an up-regulation of an Rh-like ammonia transporter in the internal tissues and excretion or storage of waste nitrogen in a so far unknown form.

The murine winged-helix transcription factor Foxl2 is required for granulosa cell differentiation and ovary maintenance.

Human Blepharophimosis/ptosis/epicanthus inversus syndrome (BPES) type I is an autosomal dominant disorder associated with premature ovarian failure (POF) caused by mutations in FOXL2, a winged-helix/forkhead domain transcription factor. Although it has been shown that FOXL2 is expressed in adult ovaries, its function during folliculogenesis is not known. Here, we show that the murine Foxl2 gene is essential for granulosa cell differentiation and ovary maintenance. In Foxl2(lacZ) homozygous mutant ovaries granulosa cells do not complete the squamous to cuboidal transition leading to the absence of secondary follicles and oocyte atresia. We further demonstrate that activin-betaA and anti-Mullerian inhibiting hormone expression is absent or strongly diminished in Foxl2(lacZ) homozygous mutant ovaries. Unexpectedly, two weeks after birth most if not all oocytes expressed Gdf9 in Foxl2(lacZ) homozygous mutant ovaries, indicating that nearly all primordial follicles have already initiated folliculogenesis at this stage. This activation, in the absence of functional granulosa cells, leads to oocyte atresia and progressive follicular depletion. In addition to providing a molecular mechanism for premature ovarian failure in BPES, these results suggest that granulosa cell function is not only crucial for oocyte growth but also to maintain follicular quiescence in vivo.

Alpha complementation in the Cre recombinase enzyme.

The Cre-loxP system is increasingly exploited for spatial and temporal gene inactivation. Here we present a novel approach to achieve this goal of selective gene inactivation. Following the model of alpha complementation in the beta-galactosidase enzyme, where the enzyme is split into independent polypeptides which are able to associate and maintain the enzymatic activity, we divided the Cre recombinase into two independent polypeptides (one containing the NH(2) terminus (alpha) and a second one containing the COOH-terminus (beta)). Individually, the two polypeptides have no detectable activity. However, when coexpressed the polypeptides are able to associate, giving rise to Cre enzymatic activity, which optimally is as high as 30% of that seen with wildtype Cre recombinase in vitro. We present this strategy as a modification of the traditional Cre-loxP system, which could be used to obtain a highly specific recombination pattern by expressing the two halves under the control of separate promoters.

ER-based double iCre fusion protein allows partial recombination in forebrain.

Here we describe the generation of a new tamoxifen-inducible double Cre fusion protein generated by fusing two ERT2 domains onto both ends of the iCre recombinase (a codon improved Cre recombinase). This Cre fusion protein (ERiCreER) had a twofold increased activity in cell culture assays than the previously described MerCreMer Cre double fusion protein. ERiCreER was targeted to the brain by placing it under the control of the promoter from the CamKIIalpha gene using a 170 kb BAC. The fusion protein was detected in hippocampus, cortex, striatum, thalamus, and hypothalamus but not in cerebellum. The ERiCreER was cytoplasmatic in the absence of tamoxifen and translocated into the nucleus upon tamoxifen administration. The activity of the ERiCreER was tested in vivo by mating the CamKIIalpha ERiCreER transgenic line with mice harbouring exon 10 of the CREB gene flanked by two LoxP sites. In the absence of tamoxifen, no background activity was detected in mice older than 6 months. After tamoxifen administration, most if not all of the ERiCreER fusion protein translocated from the cytoplasm to the nucleus; however, only 5-10% of the "floxed" CREB allele was recombined. Recombination was also visualised at the cellular level by following the upregulation of the CREM protein, which corresponds precisely with CREB loss/recombination. Unlike in other tissues (Sohal et al., 2001; Tannour-Louet et al., 2002), it appears that in brain, although ERiCreER can bind tamoxifen, the Cre-recombinase cannot be fully activated.