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.

Signaling of an allosteric, nanomolar potent, low molecular weight agonist for the follicle-stimulating hormone receptor.

Follicle-stimulating hormone (FSH) activates FSH receptors (FSHR) in granulosa cells to induce follicle differentiation, growth and estradiol production. FSH is used clinically to treat female infertility and is administered by injection. To increase patient convenience and compliance, compound homogeneity and composition, low molecular weight (LMW), orally bioavailable, FSHR agonists are now being developed to replace FSH.

Pharmacologic profiling of corifollitropin alfa, the first developed sustained follicle stimulant.

Corifollitropin alfa (Elonva®, MSD, previously N.V. Organon or Schering-Plough Oss, The Netherlands) is a newly developed sustained follicle stimulant composed of the α subunit of human follicle-stimulating hormone (FSH) and a hybrid β subunit formed by fusion of the human chorionic gonadotropin β subunit carboxy terminal peptide with the β subunit of human FSH.

Increased plasma membrane expression of human follicle-stimulating hormone receptor by a small molecule thienopyr(im)idine.

A thienopyr(im)idine (Org41841) activates the luteinizing hormone (LH) receptor but does not compete with the natural ligand binding site and does not show agonistic action on the follicle-stimulating hormone receptor (hFSHR) at sub-millimolar concentrations. When this drug is preincubated at sub-micromolar concentrations with host cells expressing the hFSHR, and then washed out, binding analysis and assessment of receptor-effector coupling show that it increases plasma membrane expression of the hFSHR. Real-time PCR shows that this effect did not result from increased hFSHR mRNA accumulation.

A tissue-selective nonsteroidal progesterone receptor modulator: 7,9-difluoro-5-(3-methylcyclohex-2-enyl)-2,2,4-trimethyl-1,2-dihydrochromeno[3,4-f]quinoline.

The progesterone receptor plays an important role in the female reproductive system. Here we describe the discovery of a new selective progesterone receptor modulator (SPRM). In rats, the lead compound, 7,9-difluoro-5-(3-methylcyclohex-2-enyl)-2,2,4-trimethyl-1,2-dihydrochromeno[3,4- f]quinoline ( 5c), inhibited ovulation and showed full efficacy in uterine and vaginal tissue but was a mixed partial agonist/antagonist in breast tissue.

Effects of the introduction of in vitro assays on the use of experimental animals in pharmacological research.

The introduction of in vitro assays in pharmacological research has led to a reduction in the number of experimental animals used. But what has been the degree of this reduction, and when did it really start? This report describes the events in a medium-sized pharmaceutical company. Analysis of data collected over the last 12 years shows a five-fold reduction in the number of experimental animals used per compound synthesised.

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.

ORG 33628 and ORG 31710 to control vaginal bleeding in progestin-only contraceptive regimens.

ORG 31710 and ORG 33628 were used in studies aiming to control vaginal bleeding in combination with progestagen-only contraception in primates. Preclinical evidence in monkeys with ORG 31710 has shown that a monthly supplementary administration to a progestagen-only contraceptive improves cycle control, probably as a result of a local endometrial effect (i.e.

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.

Effects of sea water and stanniectomy on branchial Ca(2+) handling and drinking rate in eel (Anguilla anguilla L.).

We examined the effects of seawater adaptation and extirpation of the Stannius corpuscles on branchial Ca(2+) flows, gill plasma membrane Ca(2+) transporters and drinking rate of European eels, Anguilla anguilla. Transepithelial Ca(2+) inflow in the gills increased 2 weeks after transfer of the eels from fresh water to sea water and after stanniectomy. Neither of these treatments changed the membrane density or the affinity of the Ca(2+)-extrusion mechanisms (Ca(2+)-ATPase and Na(+)/Ca(2+)-exchanger) in the gill cells, as measured in basolateral plasma membrane vesicles.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.