Report of the Federation of European Laboratory Animal Science Associations Working Group on animal identification.

The primary aim of this report is to assist scientists in selecting more reliable/suitable identification (ID) methods for their studies. This is especially true for genetically altered (GA) animals where individual identification is strictly necessary to link samples, research design and genotype. The aim of this Federation of European Laboratory Animal Science Associations working group was to provide an update of the methods used to identify rodents in different situations and to assess their implications for animal welfare. ID procedures are an indispensable prerequisite for conducting good science but the degree of invasiveness differs between the different methods; therefore, one needs to make a good ethical evaluation of the method chosen. Based on the scientific literature the advantages and disadvantages of various methods have been presented comprehensively and this report is intended as a practical guide for researchers. New upcoming methods have been included next to the traditional techniques. Ideally, an ID method should provide reliable identification, be technically easy to apply and not inflict adverse effects on animals while taking into account the type of research. There is no gold standard method because each situation is unique; however, more studies are needed to better evaluate ID systems and the desirable introduction of new and modern approaches will need to be assessed by detailed scientific evaluation.

Tibolone and metabolites induce prolactin production in human endometrial stromal cells in vitro: evidence for cell-specific metabolism.

In this study, we assessed the effects of tibolone and its metabolites on the production of a progesterone sensitive parameter, prolactin, in human endometrium stroma cells in vitro. In addition, the metabolism of the compounds by isolated stromal and epithelial cells was evaluated. The reference compounds, progesterone, Org 2058, and DHT all induced prolactin production. Oestradiol also slightly induced prolactin production and enhanced the response to Org 2058. Tibolone and Delta4-tibolone were similar with regard to potency to induce prolactin levels in the culture supernatant. Their potency was lower than that of Org 2058, similar to that of progesterone and higher than that of DHT. The efficacies of tibolone, Delta4-tibolone and Org 2058 were similar (approximately 200-fold induction). The estrogenic tibolone metabolites 3alpha- and 3beta-OH tibolone also significantly stimulated prolactin production. Their potency, however, was low since significance was reached only at the highest concentrations tested. The PR antagonist Org 31710 inhibited both tibolone- and Delta4-tibolone-induced prolactin production. The responses of tibolone and Delta4-tibolone were not affected by co-incubation with the androgen receptor antagonist OH-flutamide. The effect of tibolone, but not Delta4-tibolone, was antagonized approximately 50% in combination with the highest dose (1 microM) estrogen receptor antagonist, ICI 164384. The induction of prolactin by 3alpha- and 3beta-OH tibolone was antagonized most potently by Org 31710, but also by ICI 164384 and OH-flutamide. Tibolone is metabolized differently in epithelial and stromal cells of the human endometrium. The epithelial cells mostly produce the progestagenic/androgenic Delta4-tibolone. The stromal cells produce predominantly the 3beta-OH tibolone, and some Delta4-tibolone, but the net effect observed with regard to prolactin production is progestagenic. When the metabolites 3alpha-OH, 3beta-OH, and Delta4-tibolone were added to the cultures no conversions were observed. The HPLC analyses showed no evidence for the production of sulfated metabolites. In conclusion, the net effects on endometrial stromal cells are predominantly progestagenic. Tibolone is converted by epithelial cells into Delta4-tibolone which displays progestagenic and androgenic activities, whereas in stromal cells also the estrogenic metabolites 3alpha- and 3beta-OH tibolone are formed.

Oestrogen and progestin responses in human endometrium.

Our understanding of the mechanisms of the actions of oestrogens and progestins have evolved from the simple concept of nuclear receptor-mediated regulation of transcription to a highly sophisticated, finely tuned interplay between various coregulators, other signaling cascades and transcription factors. The net result of these complex regulatory mechanisms is a steroid-, cell-, or tissue-specific action of oestrogens and progestins, their antagonists or selective modulators of their receptors. In this review, we have attempted to shed some light on the regulation of the actions of oestrogens and progestins on the human endometrium.

Preclinical experience with two selective progesterone receptor modulators on breast and endometrium.

Org 31710 and Org 33628 are two highly selective progesterone receptor modulators (PRMs) with respect to their anti-progestational and anti-glucocorticoid activity. The compounds have been studied both in vitro and in vivo. Org 33628 has approximately four times stronger anti-progestational activity in vitro than does Org 31710, and in rats it is about 15 times more potent in the pregnancy interruption test. Two main indications for the use of PRMs are breast cancer and fertility regulation. The effects of both Org 31710 and Org 33628 were tested in relevant models for these indications. The effects of the two compounds on breast tumor development were assessed and in rats using the DMBA model. Their potency in menses induction was tested in monkeys on a 4-day regimen in the luteal phase, and after a single dose at day 21 of the normal cycle, and under a continuous progestin treatment using desogestrel. The compounds were also tested alone in a continuous low-dose regimen. The effects on follicular development and ovulation were determined by measuring estradiol and progesterone levels. Cycle control was monitored by daily vaginal swabs. In the DMBA model, Org 31710 at oral doses of 0.8, 2.0, and 5.0 mg/kg showed a clear dose-related reduction in tumor load. With the two highest doses, an even lower tumor load was seen after a 3-week treatment period compared to the tumor load at the start of treatment. Org 33628 showed a similar efficacy as Org 31710 at a dose of 2.0 mg/kg. RU 486 after oral treatment was two times less potent in this model than Org 31710 and Org 33628. The efficacy of menses induction using the 4-day regimen is dependent on the time of administration relative to the progesterone peak in the luteal phase. The highest efficacy is achieved in the descending part of the peak, at which a 100% success rate is found with a dose of 1 mg/kg of either Org 31710 or Org 33628. In Cynomolgus monkeys, at a single dose of 15 mg/kg of Org 31710 or Org 33628 in the luteal phase, menses induction was achieved only in 60% of the treatment cycles. Surprisingly menses induction can be achieved with a single dose that is about a ten-times lower when the monkeys are treated continuously with desogestrel. Cycle control is better at low than at high doses of antiprogestin in combination with daily dosing of 4 microg/kg desogestrel. Despite the difference in receptor affinity, no difference between Org 31710 and Org 33628 was found in menses induction. In the continuous low-dose (1 mg/kg) regimen with the PRMs, follicular development occurs normally while ovulation is inhibited. Ovulation is resumed shortly after stopping treatment, and a normal menses occurs after the first progesterone peak. Both compounds may be interesting options for the prevention and treatment of breast cancer and for fertility control.

Inhibition of human erythrocyte Ca2+-ATPase by Zn2+.

Recent investigations suggest that Ca2(+)-ATPase from fish gills is very sensitive to Zn2+ (Hogstrand et al., 1996. Am. J. Physiol. 270, R1141-R1147). The effect of free Zn2+ ion on the human erythrocyte plasma membrane Ca2(+)-ATPase was investigated to explore the possible extension of this finding to humans. Membrane vesicles were prepared and the Ca2(+)-ATPase activity was measured as Ca2(+)-stimulated ATP hydrolysis and as ATP-dependent Ca2+ transport. The Zn2+ ion inhibited the erythrocyte Ca2(+)-ATPase by reducing Vmax and increasing the K0.5. While in the Ca2+ transport assay only the Vmax was affected at lower Zn2+ concentrations (50-100 pM), reduction of Vmax was always accompanied by an affinity decrease in the ATP hydrolysis assay. The Ca2(+)-ATPase was found to be inhibited by Zn2+ at extremely low concentrations. The IC10 and IC50 for Zn2+, at a Ca2+ concentration of 1.0 microM, were estimated at 4 and 80 pM, respectively. Although the Ca2(+)-ATPase might be more sensitive in vitro than in vivo conditions, the results suggest that physiological concentrations of Zn2+ may reduce the activity of the erythrocyte Ca2(+)-ATPase. Furthermore, disturbance of Ca homeostasis may be a mechanism causing Zn toxicity during exposure.

Na(+)-dependent Ca2+ uptake in isolated opercular epithelium of Fundulus heteroclitus.

It is concluded that Ca2+ transport across the basolateral membranes of the ionocytes in killifish skin is mediated for the major part by a Na+/Ca(2+)-exchange mechanism that is driven by the (transmembrane) Na+ gradient established by Na+/K(+)-ATPase. The conclusion is based, firstly, on the biochemical evidence for the presence of a Na+/Ca(2+)-exchanger next to the Ca(2+)-ATPase in the basolateral membranes of killifish gill cells. Secondly, the transcellular Ca2+ uptake measured in an Ussing chamber setup was 85% and 80% reduced in freshwater (FW) and SW (SW) opercular membranes, respectively, as the Na+ gradient across the basolateral membrane was directly or indirectly (by ouabain) reduced. Thapsigargin or dibutyryl-cAMP/IBMX in SW opercular membranes reduced Ca2+ influx to 46%, comparable to the effects seen in FW membranes [reduction to 56%; Marshall et al. 1995a]. Basal Ca2+ influx across the opercular membrane was 48% lower in membranes from fish adapted to SW than in membranes from fish adapted to FW. Branchial Na+/K(+)-ATPase activity was two times higher in SW adapted fish.

Mitochondria-rich cells in gills of tilapia (Oreochromis mossambicus) adapted to fresh water or sea water: quantification by confocal laser scanning microscopy

We used confocal laser scanning microscopy to validate a new and fast co-labelling method to study the distribution of mitochondria-rich (MR) cells in gill filaments and to differentiate between MR cells that are in contact with the water (cells labelled with both DASPMI and Concanavalin-A) and those that are not (DASPMI-positive only). This method was used to describe differences in MR cell density that occur in the gills of tilapia Oreochromis mossambicus adapted to fresh water or sea water. In fresh water, the total MR cell density was 6233 cells mm-2 and the density of the subpopulation of MR cells that are in contact with the water was 3458 mm-2. After seawater adaptation, cell density decreased to 3061 cells mm-2 for all MR cells of which 2445 cells mm-2 were in contact with water. The percentage of double-labelled MR cells in the total MR cell population had increased from 55 to 80 %. MR cell size (measured as the maximal cross-sectional area) increased from 87 µm2 in fresh water to 217 µm2 in sea water. Biochemical determination of specific and total Na+/K+-ATPase activity in gill homogenates showed no difference between freshwater- and seawater-adapted fish. Quantification of 'mature' chloride cell density in fixed gill filaments using scanning electron microscopy resulted in an overestimate of chloride cell density due to shrinkage of the sample.

Mechanisms of zinc uptake in gills of freshwater rainbow trout: interplay with calcium transport.

The uptake mechanism of Zn2+ through the gill epithelium of freshwater rainbow trout was investigated both in intact animals and in isolated basolateral membranes. Involvement of the apical Ca2+ uptake sites in Zn2+ uptake was examined in vivo by pharmacological manipulation of the apical Ca2+ permeability. The apical entries of Ca2+ and Zn2+, but not Na2+ and Cl-, were inhibited by addition of La to the water. Addition of 1.0 microM La reduced the influxes of Ca2+ and Zn2+ to 22 +/- 3 and 53 +/- 7% (mean +/- SE) of the control value, respectively. Injection of CaCl2 also reduced the branchial influxes of Ca2+ and Zn2+. This treatment decreased the influx of Ca2- to 45 +/- 4% of the control level and the Zn2+ influx to 68 +/- 5%. These results strongly imply that Zn2+ passes across the apical membrane of the chloride cells of the gills via the same pathway as Ca2+. The presence of an active basolateral transporter for Zn2+ was investigated in vitro on isolated basolateral membranes. There was no ATP-dependent or Na2+(-)gradient driven transport of Zn2+ at physiological Zn2+ activities. The same system was used to study potential effects of Zn2+ on the basolateral Ca2+(-)adenosinetri-phosphatase. Zn2+ was found to be a potent blocker of this transporter, causing a mixed inhibitory effect on the ATP driven Ca2+ transport at a free Zn2+ activity of 100 pM.

Effects of Zn(2+) on Ca (2+) uptake by mitochondria and endoplasmic reticulum in permeabilized tilapia gill cells.

An intracellular ATP-dependent Ca(2+) pumping mechanism, distinct from mitochondrial Ca(2+) accumulation, was identified within tilapia gill cells. Cell suspensions treated with 0.003% saponin, which selectively permeabilizes the plasma membrane, were used to characterize the Ca(2+) sequentering mechanisms as endoplasmic reticulum and mitochondria and to determine the effect of Zn(2+) on their Ca(2+) storing activity. Of the Ca(2+) taken up by the endoplasmic reticulum, 80% was released by IP3 (10 µmol l(-1)). The Ca(2+) pump of the endoplasmic reticulum was 2.5 times less sensitive to Zn(2+) (IC50=0.05 nmol l(-1)) than was the mitochondrial uptake mechanism (IC50=0.20 nmol l(-1)). The results indicate that Ca(2+) is stored predominantly within the endoplasmic reticulum at 0.1 µmol l(-1) and that this storing capacity is seriously attenuated by namomolar concentrations Zn(2+).

N-terminal and C-terminal fragments of the hormone stanniocalcin show differential effects in eels.

The effects of an N-terminal, a C-terminal, and a mid-fragment of stanniocalcin, the primary hypocalcemic hormone in fish, on plasma total and free (ionic) calcium levels and whole animal calcium influx were tested in eels. Both the N- and the C-terminal fragments were hypocalcemic, causing 18 and 12% reduction in plasma calcium in stanniectomized eels, respectively. With both fragments the hypocalcemic action is transient. The hypocalcemia caused by the C-terminal fragment, although more rapid, is not as pronounced as the hypocalcemic action of the N-terminal fragment. Only the C-terminal fragment reduced calcium influx. The hypocalcemic activity of the C-terminal fragment then can be explained by its effect on calcium influx. The N-terminal fragment appears to function in a different manner. The mid-fragment has no effect on plasma calcium or calcium influx. The different parts of the hormone are concluded to have different effects.

A passive immunization technique against the teleost hypocalcemic hormone stanniocalcin provides evidence for the cholinergic control of stanniocalcin release and the conserved nature of the molecule.

An in vivo bioassay based on 45Ca uptake from the ambient medium was used to test the efficacy of serum from rabbits immunized against trout stanniocalcin to passively immunize trout, tilapia, American eel, and guppy against endogenous stanniocalcin. The passive immunization was effective in all species. The fact that this procedure worked under both homologous and heterologous conditions, and in fish from different taxonomic infradivisions, is consistent with the view that the stanniocalcins in the four species examined share common antigenic determinants. The trout stanniocalcin antiserum had no effect on whole body calcium uptake (inCa2+) in stanniectomized eels, indicating that the effect of the antiserum was dependent on the presence of functional Stannius corpuscles. The technique was then used to show that the inhibitory effects that calcium loading and the injection of the cholinoreceptor agonist carbachol have on inCa2+ probably involve a catecholamine-induced release of endogenous stanniocalcin from the Stannius corpuscles.

Calcium pump activities in the kidneys of Oreochromis mossambicus

The mechanism that underlies transcellular Ca2+ reabsorption in the kidney of the euryhaline teleost Oreochromis mossambicus was studied. Preparations of membrane vesicles made from the kidneys of freshwater- and seawater-adapted fish were more than sevenfold enriched in the basolateral plasma membrane marker Na+/K+-ATPase. Significant recovery of NADH­ cytochrome c reductase enzyme activity and of oxalate-stimulated Ca2+ pump activities in the membrane preparations indicated that the membrane fraction was of endoplasmic reticular origin. Indeed, thapsigargin specifically inhibited Ca2+ pump activity that could be attributed to oxalate-permeable endoplasmic reticular fragments. Kinetic analysis of thapsigargin-insensitive Ca2+ pump activity indicated the existence of a homogeneous, high-affinity, ATP-driven Ca2+ pump. No Na+-driven Ca2+ transport mechanism could be demonstrated. Plasma membrane Ca2+ pump activity was 56 % lower in preparations from seawater-adapted fish than in preparations from freshwater-adapted fish, suggesting a physiological role for this Ca2+ pump activity in renal Ca2+ handling by euryhaline species, with an involvement in the regulation of Ca2+ reabsorption.

Branchial chloride cells in larvae and juveniles of freshwater tilapia Oreochromis mossambicus

Branchial chloride cells in the developing larvae and juveniles of freshwater tilapia, Oreochromis mossambicus, were identified and the membrane Na+/K+-ATPase was localized in situ through binding of the fluorescent dye anthroylouabain. After co-labelling of the cells with the fluorescent probes DASPMI and Con-A-FITC, the mitochondria and apical crypt in the same chloride cells were visualized using confocal laser scanning microscopy. The high density of apical crypts indicated that many chloride cells were functional. The density of branchial chloride cells in larvae 10 days after hatching was approximately 6000 mm-2. An extremely high Na+/K+-ATPase specific activity of approximately 1500 µmol Pi h-1 mg-1 was measured in the gills 10 days after hatching. With the development of secondary lamellae and hence an increase in the amount of branchial epithelial protein, a concomitant decrease in the specific activity of the enzyme in the gill tissues was observed. Total Na+/K+-ATPase activity increased markedly in the early life stages. Our data indicate that in larval stages of fish the gills form a functional ionoregulatory organ before they start functioning as a gas-exchange organ.

Calcium transport in gill plasma membranes of the crab Carcinus maenas: evidence for carriers driven by ATP and a Na+ gradient.

A procedure was developed for the preparation of inside-out vesicles from plasma membranes isolated from the branchial epithelium of the green shore crab Carcinus maenas (L.). Procedures normally applied to fish branchial epithelium required the introduction of an additional hypotonic shock to obtain a preparation containing 22% inside-out vesicles, 33% right-side-out vesicles and 45% leaky membrane fragments. In such membrane preparations, the first direct evidence for uphill (against a [Ca2+] gradient) ATP-dependent and Na(+)-gradient-dependent Ca2+ transport in crustacean gills was found. The affinity for Ca2+ of the ATP-driven Ca2+ transporter was 149 nmol l-1 and that of the Na+/Ca2+ exchanger was 1.78 mumol l-1; the Vmax values were 1.73 and 9.88 nmol min-1 mg-1 protein respectively. The relative importance of these carriers for Ca2+ transport in the branchial epithelium of the crab is evaluated on the basis of their calcium kinetics.

Kinetics of ATP- and Na(+)-gradient driven Ca2+ transport in basolateral membranes from gills of freshwater- and seawater-adapted tilapia.

Plasma membranes of the gills of freshwater- and seawater-adapted tilapia were analyzed for Ca(2+)-ATPase and Na+/Ca2+ exchange activity. The relative importance of ATP-driven and Na(+)-gradient-driven Ca2+ transport in Ca2+ extrusion was evaluated on the basis of kinetic analyses in vitro. The Na+/Ca2+ exchangers in branchial membranes from freshwater or seawater fish displayed similar kinetics. The ATP-driven Ca2+ pump, however, showed a somewhat lower affinity for Ca2+ in membranes isolated from seawater gills than in membranes from freshwater gills; no difference in Vmax was found. The activity of the exchanger was estimated to be 50% of that of the ATP-driven pump at prevailing cytosolic Ca2+ concentrations (10(-7) mol l-1). Opercular ionocyte densities and branchial Na+/K(+)-ATPase content were not significantly different in fish residing in fresh water or sea water. We conclude that the gills of tilapia living for prolonged periods in fresh water or sea water do not differ in the make-up of their basolateral membrane with regard to Ca(2+)-ATPase, Na+/Ca2+ exchange and Na+/K(+)-ATPase activity. Apparently, the densities of these carriers suffice for calcium and sodium homeostasis under these vastly different ambient conditions.

A C-terminal fragment of the hormone stanniocalcin is bioactive in eels.

An in vivo eel bioassay based on 45Ca uptake from the ambient medium was used to compare the activity of native trout and American eel stanniocalcin (STC) with that of a synthetic C-terminal fragment (AA 202-231; peptide W) of Australian eel STC. Qualitatively, the action of peptide W resembled that of purified native rainbow trout (Oncorhynchus mykiss) STC as well as fresh extracts of American eel Stannius corpuscles in that they all inhibited whole animal Ca2+ influx. This inhibition was observed in both intact and stanniectomized eels regardless of whether the eels were exhibiting high or low initial calcium uptake rates. These observations suggest that peptide W activates eel gill STC receptors. Further, as peptide W was previously reported to stimulate Ca2+ influx in intact rainbow trout fry, the present data provides evidence for a difference in the way this fragment functions in various species.

Studies on stanniocalcin: characterization of bioactive and antigenic domains of the hormone.

Stanniocalcin (STC) decreases branchial Ca(2+)-uptake in fish. In order to determine its bioactive domain, synthetic fragments (U amino acids (aa) 1-20; V aa 103-136; W aa 202-231) of eel STC were tested for their effect on Ca2+ uptake in tilapia (Oreochromis mossambicus). Ca2+ uptake was inhibited by an N-terminal fragment but not by a midfragment nor a C-terminal fragment of the mature hormone. We provide theoretical and experimental evidence that a midportion of STC, which is included in the synthetic fragment V, is the most antigenic site of the molecule. Polyclonal antibodies against stanniocalcin are directed against this midportion although this region of STC appears not to be essential for signal transduction. These results suggest that the currently available antibodies will recognize inactive STC fragments in the circulation. We conclude that the bioactive portion of STC does not correspond with the major antigenic portion of the hormone. The results imply that studies on plasma STC levels employing a polyclonal antiserum against STC should be interpreted with care.

Branchial calcium uptake: possible mechanisms of control by stanniocalcin.

The branchial Ca(2+) uptake by teleost fish is under inhibitory control by the hormone stanniocalcin (STC) which is generated by the corpuscles of Stannius (CS). Removal of the CS in North American eel, Anguilla rostrata LeSueur, induced a rapid rise in blood calcium levels. Branchial Ca(2+) influx following the extirpation of the CS (stanniectomy, STX) increased during the first four days and stayed elevated thereafter (in agreement with previous studies). The transepithelial potential (TEP) across the gills did not change after STX and this means that the electrochemical gradient for Ca(2+) is less favourable for passive influx of Ca(2+) in STX eel. Therefore, the Ca(2+) influx in STX eels is a transcellular flux, with Ca(2+) crossing the apical and basolateral membrane barrier. The kinetics of ATP-driven Ca(2+)-transport across basolateral plasma membranes from eel gills did not change after STX. Thus, the increased Ca(2+)-influx after STX is not correlated with changes in ATP-dependent Ca(2+)-extrusion across the basolateral membrane, suggesting a regulation at the apical membrane. Moreover, STC did not affect ATP-driven Ca(2+)-transport in isolated basolateral membranes (in vitro). STC (0.1 nM) reduced cAMP levels in dispersed eel gill cells. It had no significant effect on the IP3 levels in these cells. We postulate that STC controls the permeability to Ca(2+) of the apical membranes of the Ca(2+) transporting cells of fish gills by controlling second messenger operated Ca(2+) channels in the apical membrane.

Inhibition of Ca2+ uptake in freshwater carp, Cyprinus carpio, during short-term exposure to aluminum.

In carp exposed to pH 5.2 in fresh water, the Ca2+ influx from the water is reduced by 31% when compared to fish in water of neutral pH. At pH 5.2, the Ca2+ influx but not Na+ uptake is decreased by aluminum (Al). Al reduces Ca2+ influx dose-dependently: a maximum 55% reduction was observed after 1-2 h exposure to 200 micrograms.1(-1) (7.4 microM) Al. Branchial Ca2+ efflux is less sensitive to Al and affected only by exposure for more than 1 h to high Al concentrations. Na+ influx is not affected by concentrations Al up to 400 micrograms.1(-1). Na+ efflux, similarly to Ca2+ efflux, increased when fish were exposed for more than 1 h to 400 micrograms.1(-1) Al.

Cadmium inhibition of the erythrocyte Ca2+ pump. A molecular interpretation.

The effects of cadmium (Cd2+) on transmembrane Ca2+ transport and on the membrane permeability for Ca2+ were studied in human erythrocytes. The erythrocyte Ca2+ pump is inhibited competitively by Cd2+ via interaction with the Ca2+ transport site of the carrier and not via interaction with its activator calmodulin. The affinity of the Ca2+ pump for Cd2+ is extremely high (KI = 2.0 nM Cd2+). Cd2+ (less than or equal to 10(-4) M) does not alter the membrane permeability for Ca2+. We conclude that the pivotal mechanism in the toxic action of Cd2+ is the inhibition of Ca2+-ATPase mediated Ca2+ extrusion. As a result Cd2+ disturbs intracellular Ca2+ homeostasis and may increase cytosolic Ca2+ (Ca2+i) to toxic levels.