th Recent Advances in Proceedings of 4 Congress of the Asia and Comparative Endocrinology Oceania Society for Comparative Endocrinology, (Editor: J.Y.L. Yu), 2001 May 14-18, 2000, Taipei, Taiwan, R.O.C.
UNIHI-SEAGRANT-CR-00-01
HORMONAL CONTROL OF OSMOREGULATION IN A STENOHALINE CATFISH, ICTALURUS PUNCTATUS
Steve M. Eckert, Tetsuya Hirano and E. Gordon Grau
Hawaii Institute of Marine Biology, Universityof Hawaii, P. 0. Box 1346, Coconut Island, Kaneohe, HI 967 44, USA
SUMMARY
When channel catfish (lchtalurus punctatus) in freshwater (FW) were hypophysectomized, plasma osmolality decreased significantly after 24 hrs, and the maximum reduction was seen after 2 days. When they were transferred from FW to dilute seawater (20-40% SW), plasma osmolality of the sham-operated fish was consistently higher than the osmolality of the environmental water, whereas the osmolality of the hypophysectomized fish was equivalent to the environmental salinity. Ovine prolactin (PRL) restored the reduced osmolality of the hypophysectomized fish in FW to the level of sham-operated fish. Cortisol was also effective, but the effect was less pronounced than the effect of PRL. Injection of PRL in combination with cortisol resulted in a marked increase in plasma osmolalityto a level even above that of the sham-operated fish. Ovine growth hormone (GH) was without effect. Replacement therapy in the hypophysectomized fishtransferred to dilute SW revealed essentially the same results as in the fish in FW. In the stenohaline catfish, both PRL and cortisol seem to be involved importantly in the ion uptake from the environment not only in FW but also in a saline environment.
INTRODUCTION
It has been accepted generally that prolactin (PRL) is the dominant factor ih regulating hydromineral balance in fresh water and that cortisol is the dominant factor in seawater (1-3). Recently, growth hormone (GH) and its mediator, insulin-like growth factor-I(IGF-I), have also been implicated in the control of seawater adaptation in several salmonids as well as euryhaline tilapia, Oreochromis mossambicus, and killifish, Fundulus heteroclitus (4-6). By contrast, limited informationis available on the hormonal control of osmoregulation in stenohaline fishes, although the important role of PRL has been well-established also in stenohaline freshwater species (I). The channel catfish (Ictalurus punctatus) is native to the Mississippi River drainage. They are also found in coastal waters, and successfully cultured in brackish-water ponds. The upper
- 347 - limit for their survival seems to be about 30% SW (7). Davis and Simco (8) reported that cortisol injections had no effect on increased plasma Cl and osmolality in channel catfish transferred to hyper-ionic salt water. In the Indian catfish (Heteropneustes fossilis), Parwez and Goswami (9) reported that PRL and cortisol restored decreased plasma osmolality and sodium levels of the hypophysectomized fish in fresh water. The present study was undertaken to clarify the roles of PRL, GH and cortisol in the maintenance of hydromineral balance in the channel catfish.
MATERIALS AND METHODS
Fry of channel catfish (Ictalurus punctatus) were obtained from Long View Ranch, Wetumka, OK. They were reared in circular 6000-liter tanks with fresh municipal water, and held under natural photoperiod at the Hawaii Institute of Marine Biology, University of Hawaii. They were fed Purina Trout Chow twice daily. Water temperature was 24 2°C. Fish weighing 80-120 g were randomly assigned to oval 60-liter tanks containing fresh± water or dilute seawater, that was mechanically and chemically filteredand UV sterilized. For hypophysectomy, tl!e fish were anesthetized in buffered tricaine methansulphonate (MS222, Sigma, 0.15 g/l, 0.2 g/l sodium bicarbonate). Their mouths were kept open by a retractor. A median sagittal incision (about 5 mm long) was made 'in the mucus membrane just anterior to the pair of pharyngeal teeth. The mucous membrane was retracted with a cotton-tipped applicator to expose the cranium. The pituitary is visible as a white dot through the parasphenoid. The bone ventral to the pituitary was carefully drilled with a dental drill, and the exposed pituitary was removed by suction with a glass pipette. The cranium was filled with microfibrillar collagen hemostat. The operated fish were returned to freshwater aquaria. There was usually no mortality afterthe operation. Ovine PRL (NIK-oPRL-21) and ovine GH (NIDDK-oGH-15) were a gift from Dr. A. F. Parlow, National Hormone & Pituitary Program. Cortisol was purchased form Sigma (St. Louis, MO). Hormones were suspended in vegetable oil, and injected intraperitoneally. Fish injected with vegetable oil served as a control. Blood was taken by caudal puncture using a syringe treated with ammonium heparin. After centrifugation at 10,000 rpm for 5 min, plasma was removed and stored at -80°C. Plasma osmolality was measured using a vapor pressure osmometer (Wescor 5500, Logan, UT). All values represent means standard errors of the means (SEM). Significance of differences between the two groups± was analyzed by analysis of variance (ANOVA) followed ' by Duncan's multiple-range test or Mann-Whitney U-test.
RESULTS
When channel catfish in' fresh water were hypophysectomized, plasma osmolality
- 348 - decreased significantly after 24 hrs, and the maximum reduction was observed after 2 days. All the hypophysectomized fish survived for more than 7 days. Two days after hypophysectomy, they were injected either with ovine GH (5 µgig), ovine PRL (5 µgig), cortisol (50 µgig) or ovine PRL plus cortisol (5 µgig PRL and 50 µgig cortisol). Serial blood samples were taken immediately after hypophysectomy (day -2), at the time of hormone \ injection (day 0), and 2, 4 and 7 days after injection. As is shown in Fig. I, PRLrestored the reduced osmolality to the level of sham-operated fish. Cortisol was also effective, but the effect was less pronounced than the effect of PRL. Most striking was the synergistic effect of PRL and cortisol; the plasma osmolality increased above that of the sham-operated fish. GH was without effect.
Day-2 DayO Day2 Day4 (H)ll Cllh)Rctom,y) (btjedion)
Fig. Effect of hypophysectomy of the channel catfish in FW and replacement therapy with ovine GH and cortisol (F). Mean SEM (n=-5-6). *Significantly different from the 1. corresponding hypophysectomized fish injected with vegetable oil (HX). PRL, ±
ln the next experiment, catfish in fresh water were hypophysectomized and transferred to 20% SW (200 mOsm). After 2 days, they were injected either with ovine GH (5 µgig), ovine PRL (5 µgig), cortisol (50 µgig) or ovine PRL plus cortisol (5 µgig PRL and 50 µgig cortisol). Sham-operated fish and hypophysectomized fish receiving oil served as controls. Immediately after the hormone injection, salinity of the aquaria was increased to 300 mOsm (30% SW), and increased further to 35% SW 4 days after the hormone injection. Serial blood
- �49 - samples were taken immediately after hypophysectomy (day -2), at the time of hormone injection (day 0), and 2, 4 and 6 days after injection (Fig. 2). Plasma osmolality of the sham-operated fish was consistently higher that the osmolality of the environment, whereas that of hypophysectomized fish injected with oil and ovine GH was significantly less than that of the sham-operated fish and was almost equivalent with the osmolality of the environment. As in the case of hypophysectomy and replacement therapy in fresh water, PRLrestored the reduced osmolality to the level of sham-operated fish. Cortisol was also effective, but the effect was less pronounced than the effect of PRL. The synergistic effect of PRL and cortisol was also apparent: the plasma osmolality increased above that of the sham-operated fish.
42 0 � 1 I D hm D � I � I � HX CH F PJL BIL+F 01
04 I 38>mi f :j J j
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n.� DQ-0 DQ-4 ��) (himDV FW-31n0m DJ-JD nLkm
Fig. 2. Effect of hypophysectomy of the channel catfish and replacement therapy with ovine PRL, GH and cortisol (F) during the course of acclimation to 30-35% SW. Mean SEM (n=6). *Significantly different from the corresponding hypophysectomized fish injected with ± vegetable oil (HX).
DISCUSSION
Hypophysectomy resulted in a significantreduction in plasma osmolality in channel catfish
- 350 - in FW within 24 hrs. In the Indian catfish, hypophysectomy also resulted in a marked decrease in urine flow rate, primarily due to reduced glomerular filtration rate (GFR). Urine osmolality and sodium concentration increased due to reduced tubular reabsorption of sodium (9). In addition to the reduced ability of the kidney to eliminate sufficient water, the reduction in plasma osmolality in the hypophysectomized catfishin FW is apparently due to reduced influx and/or increased loss of ions from the body surfaces, especially from the gills. On the other hand, when channel catfishwere hypophysectomized in FW and then transferredto 30-35% SW, plasma osmolality of the hypophysectomized fish increased in parallel with the environmental osmolality, while osmolality of the sham-operated fish was always kept higher than the environment. Hypophysectomized fish may behave like an osmoconformer in saline environment, possibly due to reduced ion uptake fromthe gills. There is overwhelming experimental support for the role of PRL in promoting ion uptake not only in fishes in FW (1-3). Chidamram et al. (10) reported that treatment of hypophysectomized black bullhead (1 me/as) with ovine PRL restored plasma Na to the level of intact fish. Similar effects of PRL have been observed in Indian catfish (H fossilis) after hypophysectomy in FW (9). In the Indian catfish, PRL also restored the decreased urine flow rate and GFR and attenuated the increased urine osmolality and sodium concentrations. In the present experiment, injections of ovine PRL into the hypophysectomized fish in FW restored the reduced plasma osmolality to the sham-operated level. When channel catfish were hypophysectomized and transferred to 30-35% SW, ovine PRL elevated the osmolality to the level of sham-operated fish. PRL is likely to stimulate ion uptake by the gills and free water clearance by the kidney, thus resulting in increases in plasma ion concentrations and osmolality. Several lines of evidence indicate a role for cortisol in SW acclimation of euryhaline teleosts (1-3). In the present study, however, no hyposmotic effect of cortisol was seen in the hypophysectomized catfish during the course of acclimation to dilute SW. On the other hand, the GH/IGF-I axis has been shown to play an important role in seawater adaptation of salmonids (3, 4). When injected into the fish prior to or during acclimation to SW, GH and IGF-I are known to attenuate the increase in plasma ions or osmolality. Injection of GH also increases hyposmoregulatory ability in tilapia and killifish (5, 6, 11). In the present study, however, no hyposmot1c effect of GH or cortisol was observed. SW-adapting effects of GH/IGF-I axis and cortisol may be limited to euryhaline species. On the other hand, there is an increasing body of evidence that cortisol is also involved in ion uptake in fresh water (12). Increases in plasma osmolality of hypophysectomized fishes in FW have been observed after cortisol treatment in the European eel, goldfish and Indian catfish (9, 13, 14). In accord with these observations, cortisol treatment of the hypophysectomized channel catfishrestored the plasma osmolality both in FW and dilute SW, although the effect was Jess pronounced than PRL's effect. In the Indian catfish, cortisol evoked significant increases in urine osmolality and sodium concehtration as well as urine flow rate, thus leading to marked natriuresis. As in the case of
- 351 - PRL's action, cortisol seems to increase plasma osmolality by stimulating ion uptake by the gills of catfish both in fresh water and dilute seawater, in spite of severe natriuresis. The most striking finding in the present study was the synergistic effect of PRL and cortisol in increasing plasma osmolality even above the level of sham-operated fish both in FW and in dilute SW. In the Indian catfish, Parwez and Goswami (9) reported that simultaneous administration of ovine PRL and cortisol elevated plasma osmolality and sodium levels in the hypophysectomized fish in FW to a normal level, although urine flow rate, GFR and fractional tubular reabsorption of water were still lower than in the intact control. In the present study, PRL did not evoke any increase in plasma cortisol in the hypophysectomized channel catfish either in FW or in dilute SW (data not shown). According to Parwez and Goswami (9), administration of PRL and cortisol had no effect on plasma osmolality when the fish were maintained in deionized water, indicating that these hormones increase plasma _osmolality by stimulating active uptake of ions fromthe externalenvironment. They suggested that PRL and cortisol act through independent mechanisms. Obviously, more work is required to clarify the mechanisms by which PRL and cortisol are acting on ion uptake, and the pathways through which cortisol and PRL interact in the fish in FW.
ACKNOWLEDGEMENTS
We are grateful to Prof. Milton Stetson, University. of Delaware, and Dr. N. Harold Richman and Mr. Steven Shimada, Hawaii Institute of Mar.ine Biology, University of Hawaii, and Dr. Bryan S. Shepherd, University of Kentucky, for their invaluable suggestions and encouragement during the course of this study. This study was supported by a grant fromState of Hawaii, DLNR 40402 and also by NOAA Sea Grant # NA86RG0041 and USDA grant # 9835206644.
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