Integumental and branchial chloride cells of tilapia larvae (Oreochromis mossambicus) were studied at the light-microscopical and ultrastructural level. Total numbers and distribution of chloride cells were quantified after immunostaining of cross sections of the entire larvae with an antibody against the alpha-subunit of Na+/K+-ATPase. The majority (66%) of Na+/K+-ATPase-immunoreactive (ir) cells, i.e. chloride cells, of freshwater tilapia larvae were located extrabranchially up to 48 h after hatching. Five days after hatching, the majority (80%) of chloride cells were found in the buccal cavity. Transfer of 24-h-old larvae to 20% sea water speeded up this process; 24 h after transfer (i.e. 48 h after hatching), the majority (59%) of chloride cells were located in the buccal cavity. The branchial chloride cell population of 24-h- and 120-h-old larvae consisted of immature, mature, apoptotic and necrotic chloride cells. However, relatively more immature chloride cells were observed in freshwater larvae (42-63%) than in (previously studied) freshwater adults (21%), illustrating the developmental state of the gills. After transfer to sea water, the incidence of degenerative chloride cells did not change. Furthermore, the incidence of immature cells had decreased and a new subtype of chloride cells, the "mitochondria-poor" cells, appeared more frequently. These mitochondria-poor chloride cells were characterised by an abundant tubular system and relatively few mitochondria, which were aligned at the border or concentrated in one part of the cytoplasm. Most of these cells did not contact the water. The function of their enhanced appearance after seawater transfer is unknown.
Fertile life of oocytes is usually considered to be related to ovulation time. In the present study, fertile life of rat oocytes was studied in relation to resumption of meiosis. In pro-oestrus, meiotic resumption without concomitant ovulation was induced in most graafian follicles by injection of a small amount of LH or FSH followed by Nembutal. These follicles either ovulated or developed into luteinized unruptured follicles if Ovalyse (a GnRH analogue) was given 8 h after LH or FSH. In subsequent experiments, rats were injected with FSH or saline, and Nembutal; 4 or 8 h later, Ovalyse was given to induce ovulation; the rats were mated 14 h after Ovalyse. At day 20 of pregnancy, fetal survival was 30% in rats with meiosis advanced by 8 h, against 91 % and 70% in rats advanced by 0 or 4 h, respectively. Mortality occurred mainly during pre-implantation and early post-implantation. Advanced resumption of meiosis may cause pre-ovulatory ageing of oocytes; consequently, viability of these oocytes after ovulation is reduced.
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.
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. We conclude that the increase in the Ca(2+)-transporting capacity observed in the gills of fish exposed to the larger transepithelial Ca(2+) fluxes, resulting from exposure to sea water or stanniectomy, involves an increase in number and/or size of the Ca(2+)-transporting cells, but not in the membrane density of Ca(2+) transporters. Branchial Ca(2+) outflow was higher in stanniectomised than in sham-operated fish. Changes in electrochemical driving forces as well as plasma stanniocalcin or teleocalcin levels may be the basis for the observed differences. Stanniectomy enhanced drinking in stanniectomised eels. Drinking was not further affected by transfer to sea water. These observations suggest that the corpuscles of Stannius are involved in the water balance.
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.
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