Fish Physiology and Biochemistry vol. 8 no. 2 pp 159-165 (1990) Kugler Publications, Amsterdam/Berkeley
Growth hormone secretion during longterm incubation of the pituitary of the Japanese eel, Anguilla japonica Reiko Suzuki, Mitsuyo Kishida and Tetsuya Hirano Ocean Research Institute, University of Tokyo, Nakano, Tokyo 164, Japan Keywords: Anguilla japonica, growth hormone, hypothalamic control, osmotic effect, somatostatin
Abstract Growth hormone (GH) secretion from organ-cultured pituitaries of the eel (Anguillajaponica) was studied during incubation in a defined medium for 2 weeks, using a homologous radioimmunoassay which does not distinguish between the two molecular forms of eel GH. The total amount of GH secreted increased gradually during the incubation period; so that the amount of GH released on day 14 was about 30 times greater than that on day 1. On day 14, the proportion of GH released relative to the total amount of GH present (the sum of GH released into the medium and residual content in the pituitary) was 96% and the amount produced on day 14 was 4 times greater than the content in the unincubated pituitary. Somatostatin (SRIF, 1.8 x 10- 7 M) inhibited the increase in GH release. On day 7, the proportion of GH released by pituitaries treated with SRIF (28%) was less than that released by the control pituitary (91 %). There was no significant difference in GH release between the pituitaries incubated in isotonic medium (300 mOsm) and those in hypotonic medium (240 mOsm) for 2 weeks except for the first 3 days, when the pituitaries in hypotonic medium secreted significantly greater amounts of GH than those incubated under isotonic condition. Hypertonic medium (350 mOsm) had no effect on GH release except for significant inhibition on days 6 and 14. When secretion of the two forms of GH (GH I and II) was examined after separation by polyacrylamide gel electrophoresis followed by densitometry, slightly more GH I tended to be secreted than GH II during the culture period, although the effects of SRIF and osmolality of the media on GH I release were similar to those on GH II. It is concluded that GH secretion and production in the eel is mainly under the inhibitory control of hypothalamus, and that osmolality has a minimum influence on the GH release.
Introduction There is increasing evidence suggesting that growth hormone (GH) plays an important role in seawater adaptation of salmonid fishes (Komourdjian et al. 1976; Clarke et al. 1977; Miwa and Inui 1985; Bolton et al. 1987), and an increase in plasma GH levels has been reported in juvenile chum salmon trans-
ferred from fresh water to seawater (Hasegawa et al. 1987; Hirano et al. 1987; Ogasawara et al. 1988). According to Nishioka et al. (1987), hypophysectomized coho salmon are unable to survive in seawater, although they adapt well to fresh water. In tilapia, Helms et al. (1987) reported that hypertonic medium stimulates in vitro release of GH from the pituitary. In contrast, Olivereau and Ball (1970)
Address correspondence to: Reiko Suzuki, Ocean Research Institute, Universily of Tokyo, Nakano, Tokyo 164, Japan.
160 reported a slight reduction in the activity of the GH cells after transfer of the European eel (Anguilla anguilla)from fresh water to seawater, whereas an activation was seen after transfer from fresh water to de-ionized water (Olivereau 1967). GH release from the eel (A. anguilla) pituitary in vitro has been reported to be less in high sodium medium than in low sodium medium (Baker and Ingleton 1975). The hypothalamic control of GH secretion in teleosts has received little attention, although it is suggested to be predominantly under stimulatory control (Ball et al. 1972; Ball 1981). In the eel, GH cells in homotransplanted pituitaries were moderately active histologically (Olivereau 1970). According to Baker and Ingleton (1975), organ-cultured eel pituitaries continued to secrete GH, estimated by densitometry after gel electrophoresis for 2 weeks, indicating autonomy of GH secretion. Thus, hypothalamic control as well as the response to environmental salinity of GH release in the eel would seem to be different from the other teleosts. Recently, two forms of GH have been isolated and fully characterized in the Japanese eel, A. japonica (Kishida et al. 1987; Yamaguchi et al. 1987). A homologous and specific radioimmunoassay (RIA) which does not distinguish between the two forms has also been established recently (Kishida and Hirano 1988). The present study was undertaken to examine the effects of osmolality of the culture media and of somatostatin on GH release, using RIA for measurement of total levels of eel GHs, during incubation in a defined medium for 2 weeks. Secretion of the two GHs was also examined by densitometry after gel electrophoresis.
Materials and methods Cultured immature Japanese eel (Anguilla japonica), weighing 150 to 200 g, were maintained in a freshwater pond. After decapitation, the pituitaries were removed aseptically and preincubated for 1-2h in isotonic medium (Eagle's Minimum Essencial Medium with Earle's salts, 300 mOsm/kg H 20), containing 200 U/ml penicillin, 200 U/ml streptomycin and 0.5 /ag/ml Fungizone (M. A. Bioproducts, Walkersville MD). Two pituitaries were
cultured together for 1-2 weeks at 180C under 95% 02/5070 CO 2 in 0.22 ml medium using a multiwell dish (well diameter 6.4 mm, depth 10 mm). The culture medium contained half the concentrations of the antibiotics used for preincubation. The pH of the medium was adjusted to 7.3-7.4 with sodium bicarbonate. The medium was changed daily. At the end of the culture period, the pituitaries were homogenized in 0.2 ml distilled water. The medium and pituitary extracts were stored at -80°C until hormone assay. The hypotonic medium (240 mOsm) was prepared by diluting the isotonic medium with distilled water, and the hypertonic medium (350 mOsm) by adding mannitol. The effects of medium osmolality were examined using the pituitaries from the same stock of the eel. Somatostatin (SRIF; Peptide Institute, Osaka) was initially dissolved in distilled water and the stock solution was diluted in isotonic medium to 1.8 x 10- 7 M (300 ng/ml). Total contents of the two GHs in the culture medium and the pituitary extract were measured by a homologous RIA using rabbit antiserum against GH I which does not distinguish between two forms of the eel GH (Kishida and Hirano 1988). The culture medium (0.2 ml) and pituitary extract (0.2 ml) were also subjected to polyacrylamide disc electrophoresis at pH 9.5, as described by Davis (1964). Ovine prolactin (NIAMDD-o-PRL-14) was electrophoresed simultaneously as a reference standard. The gel was stained with 0.25% Coomassie brilliant blue. After destaining, the density of protein bands was measured using a densitometer. The bands with Rf 0.29 and 0.36 were identified as GH II and I, respectively (Kishida et al. 1987). The GH contents were expressed as zg ovine PRL equivalent. Immunocytochemical staining of Bouin-fixed, wax-embedded pituitaries was carried out according to the peroxidase-antiperoxidase (PAP) method as described by Naito et al. (1983). Sections of an unincubated pituitary and the pituitary after the culture were stained with the same antiserum as used for RIA. All data were analyzed by Duncan's new multiple range test at the 0.05 level of significance.
161
1
a
2
a
,_
Control
'3' -0t .L c
Z0
En a
0.5
0.5-
la
1
OC
0 a) 0) a
I;
I0)
.
id a, M
.(
I
0 l
1
|
z
3
7
0
ra
days -
1
2 3
6
10
12
14
days
Fig. 1. Secretion of GH from the eel pituitary into hypotonic ( o , 240 mOsm), isotonic ( , 300 mOsm) or hypertonic ( o, 350 mOsm) medium during 14 days, as measured by RIA. GH content in the unincubated pituitary and that remaining after culture are also indicated. Vertical bars indicate standard errors of the means (n = 4-6). * Significantly (p < 0.05) different from the corresponding value in isotonic medium.
Fig. 2. Effect of somatostatin ( , SRIF; 1.8 x 10- 7 M) under isotonic conditions on GH release and on residual GH content in the pituitary, as measured by RIA. Vertical bars indicate standard errors of the means (n = 5-6). * Significantly different (p < 0.05) from the control value in isotonic medium ( A).
7, the relative amount of GH released as a percentage of the total GH (sum of released and residual content) was less in the pituitary treated with SRIF (28%) than in the control pituitary (91 °7).
Results Release of total GH measured by RIA When eel pituitaries were cultured in isotonic medium, GH secretion increased gradually over 2 weeks; the amount of GH release on day 14 was approximately 30 times greater than that on day 1 (Fig. 1). The amount secreted on day 14 was about 4 times greater than the content in the unincubated pituitary and 20 times greater than the residual GH content. The hypotonic medium significantly stimulated GH release for the first 3 days, whereas no effect was seen during the rest of the experiment. Hypertonic medium had no significant effect on GH release except for an inhibition on days 6 and 14. Residual GH contents in the pituitary after culture were significantly less in hypotonic and in hypertonic media than in isotonic medium (Fig. 1). Somatostatin (SRIF, 1.8 x 10- 7 M) inhibited the rise in GH release over 7 days, and the residual GH content of the gland after 7 days was significantly greater than the control level (Fig. 2). On day
Release of two forms of GH separately measured by densitometry The release of both GHs in the isotonic medium increased gradually over 14 days, the increase in GH I secretion being slightly more than GH II (Fig. 3). Altered medium composition had no effect on the release of either form of the hormone except for an inhibition by hypertonic medium on day 14 (Fig. 3). SRIF appeared to inhibit the increase of both GHs but no statistically significant difference was observed using densitometric measurements (Fig. 4). There was no significant difference in the residual hormones in the pituitary after SRIF treatment or altered medium osmolality.
Immunocytochemistry Immunocytochemical examination of the pituitary after culture for 14 days showed that numerous
162 3
a
2
I
2
2
t IFr
-r
a,
M 0, 0, RA
_L i MZ
(3
Cr
0I A v
days
1
3
d days
a -3
3
p2 a'
2 2
I
-3 =-r 1
6
I
I
,
i a
I.
Er N v
g
CT
1
3
6
10
-0
14 days
Fig. 3. Secretion of two forms of GH from the eel pituitary into hypotonic ( o, 240 mOsm), isotonic ( , 300 mOsm) or hypertonic ( o , 350 mOsm) medium during 14 days. GH content in the unincubated pituitary and that remaining after culture are indicated as zg o-PRL equivalent/pituitary. Values were measured by densitometry after gel electrophoresis. Vertical bars indicate standard errors of the means (n = 4-6). * Significantly (p < 0.05) different from the corresponding value in isotonic medium.
immunoreactive GH cells were found in the cultured pituitary, as in the unincubated pituitary (Fig. 5).
Discussion In this study, organ-cultured eel pituitaries secreted gradually increasing amounts of GH over 2 weeks, and the amount of GH present on day 14 (a sum of the amount released into the medium and the residual content in the pituitary) was 4 times greater than the GH content in the unincubated pituitary. Immunocytochemical examination showed that the pituitary after culture for 2 weeks contained as many immunoreactive GH cells as in the unincubated pituitary. Baker and Ingleton (1975) also reported an increase in GH secretion in the European eel
I5
V
n
irao
-o
3 days
Fig. 4. Effect of somatostatin ( , SRIF; 1.8 x 10- 7 M) under isotonic condition on release of GH I and II and on residual content of two forms of GH in the pituitary, as measured by densitometry after gel electrophoresis. Vertical bars indicate standard errors of the means (n = 4-6).
during the first week of culture, although a decline occurred gradually thereafter. These results indicate that the cultured pituitary are viable, and synthesize and secrete an increasing amount of GH during the culture period in a defined medium without supplements such as growth factors, hormones, or fetal calf serum etc. By contrast, the in vitro secretion of GH from the pituitaries of tilapia, rainbow trout and the mature chum salmon decreased gradually during several days of the culture, with considerable amounts of the hormones remaining in the pituitary (Baker and Ingleton 1975; Suzuki and Hirano 1987; Suzuki et al. 1987). Both production and secretion of GH in the eel pituitary seem to be normally under tonic inhibitory control, most likely by the hypothalamus, although presence of potential inhibitory factors in the blood is also possible. This seems to be in agreement with extremely low levels of GH in the eel plasma (less than 0.2 ng/ml; Kishida and Hirano 1988), although this could also be due to an extremely high metabolic clearance of the hormone.
163
Fig. 5. Sagittal section of an eel pituitary cultured for 14 days under isotonic condition (A) and of an unincubated pituitary (B), stained with an anti-eel GH rabbit serum at a dilution of 1:4000. Sections were counterstained with Mayer's hematoxylin. x 60.
In the eel, SRIF fibers terminate on the basal lamina along GH cells, and changes in SRIF immunoreactivity have been observed after starvation suggesting that SRIF regulates GH release in the eel (Olivereau et al. 1984). In the present study, SRIF significantly reduced GH release by day 7, so that residual GH in the pituitary after the culture was greater than in the control pituitary. In contrast, Hall and Chadwick (1978, 1983) reported that hypothalamic extracts stimulated GH release in vitro from the European eel pituitary. In our preliminary study, the eel pituitary incubated with a hypothalamic extract secreted less GH during the 7 day-incubation than the pituitary with an olfactory lobe extract, although the pituitary continued to secrete increasing amounts of GH for 7 days even with the hypothalamic extract (unpublished observation). Thus, GH secretion from the eel pituitary seems to be under both inhibitory and stimulatory control by the hypothalamus, with the inhibitory control being more important.
Secretion of GH from the eel pituitary was not significantly influenced by either hypotonicity or hypertonicity of the culture medium at least over a prolonged period. Recent studies using a perifusion technique revealed that reduced osmotic pressure stimulated PRL release from the tilapia pituitary only for a few hours (Grau et al. 1986, 1987) and from the rainbow trout pituitary for less than 1 hour (Gonnet et al. 1988). Gonnet et al. (1988) suggested that the temporal increase in the hormone secretion by hypotonic medium may be a result of physical modification of the outer cell membrane and not necessarily specific to PRL cells. It is highly probable, therefore, that apparent stimulation of GH release by hypotonic medium for the first 3 days may also be due to mechanical modification. It would be interesting to examine if eel GH has similar osmoregulatory effects in the eel, a catadromous fish, to those seen in the anadromous salmonids, in which GH seems to favor their seawater adaptation (see Bolton et al. 1987; Hirano et al. 1987). The eel GH RIA is more sensitive than the densitometric method; the minimum detectable levels of GH were 10 pg in the RIA (Kishida and Hirano 1988) and 1.5 + 0.3 tAgovine PRL (mean + SEM, n = 6) in the densitometry. Furthermore, interassay coefficients of variation, calculated using the midrange of the assay, were 13.8% (n = 4) in the RIA and 17.7% (n = 3) in the densitometry. These differences in sensitivity and accuracy seem to be a part of the causes for the apparent low degree of correlation between the pattern of GH release measured by RIA and that by the densitometry. For example, a statistically significant decrease in the amount of GH release, as measured by RIA, from the SRIF-treated pituitaries was observed on day 7 (Fig. 2), whereas no significant difference was seen when the densitometry was used to measure the levels of eel GH I and II (Fig. 4). The densitometry seems to be less accurate than the RIA to measure GH amounts in the pituitary, since the pituitary extracts contained proteins with isoelectric points close to those of GH I and II, resulting in increased background staining. Therefore, the residual GH content in the control pituitary as measured by RIA was significantly less than in the SRIF-treated
164 group, whereas no difference was observed by the densitometry (Figs. 2 and 4). Thus, the RIA method seems to provide a 'true' measure of the release of total amount of eel GH. Since the antiserum used for the RIA does not distinguish between the two forms of eel GH, the densitometry following electrophoresis will provide informations on the release of GH I and II separately. Eel GH I has an amino-terminal extension of three amino acid residues, compared with eel GH II, and there is no difference between the growth promoting activities of GH I and II when tested in the juvenile rainbow trout (Kishida et al. 1987; Yamaguchi et al. 1987). In the present study, slightly more GH I tended to be secreted than GH II under the all different culture conditions, although GH I and II were stored in approximately equal quantity both in the unincubated pituitary and in the pituitary after the culture. Further studies are needed to clarify the origin and the physiological role of two forms of eel GH.
Acknowledgements We express our gratitude to Dr. T. Ogasawara and Ms. S. Hasegawa, Ocean Research Institute, University of Tokyo, for their continued interest and encouragement throughout the course of this study, to Professor K. Kohmoto and Dr. S. Sakai, Faculty of Agriculture, University of Tokyo, for their help in the densitometry. We are also grateful to Professor H.A. Bern, University of California, Berkeley, and Dr. B.I. Baker, University of Bath, for their encouragements and suggestions and also for critical readings of the manuscript. This study was aided in part by grants-in-aid for scientific research from the Ministry of Education, and Fisheries Agency.
References cited Baker, B.I. and Ingleton, P.M. 1975. Secretion of prolactin and growth hormone by teleost pituitaries in vitro. II. Effect of salt concentration during long-term organ culture. J. Comp. Physiol. 100:269-282. Ball, J.N. 1981. Hypothalamic control of the pars distalis in fishes, amphibians, and reptiles. Gen. Comp. Endocrinol. 44:135-170.
Ball, J.N., Baker, B.I., Olivereau, M. and Peter, R.E. 1972. Investigations on hypothalamic control of adenohypophysial functions in teleost fishes. Gen. Comp. Endocrinol., Suppl. 3:11-21. Bolton, J.P., Collie, N.L., Kawauchi, H. and Hirano, T. 1987. Osmoregulatory actions of growth hormone in rainbow trout (Salmo gairdneri). J. Endocrinol. 112:63-68. Clarke, W.C., Farmer, S.W. and Hartwell, K.M. 1977. Effect of teleost pituitary growth hormone on growth of Tilapia mossambica and on growth and seawater adaptation of sockeye salmon (Oncorhynchus nerka). Gen. Comp. Endocrinol. 33:174-178. Davis, B.J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, Art. 2:404-427. Gonnet, F., Prunet, P., Tonon, M.C., Dubourg, P., Kah, O. and Vaudry, H. 1988. Effect of osmotic pressure on prolactin release in rainbow trout: in vitro studies. Gen. Comp. Endocrinol. 69:252-261. Grau, E.G., Ford, C.A., Helms, L.M.H., Shimoda, S.K. and Cooke, I.M. 1987. Somatostatin and altered medium osmotic pressure elicit rapid changes in prolactin release from the rostral pars distalis of the tilapia, Oreochromismossambicus, in vitro. Gen. Comp. Endocrinol. 65:12-18. Grau, E.G., Shimoda, S.K., Ford, C.A., Helms, L.M.H., Cooke, I.M. and Pang, P.K.T. 1986. The role of calcium in prolactin release from the pituitary of a teleost fish in vitro. Endocrinology 119:2848-2855. Hall, T.R. and Chadwick, A. 1978. Control of prolactin and growth hormone secretion in the eel Anguilla anguilla. Gen. Comp. Endocrinol. 36:388-395. Hall, T.R. and Chadwick, A. 1983. The effect of thyrotrophin releasing hormone on secretion of prolactin and growth hormone from eel pituitaries incubated in vitro. IRCS Med. Sci. 11:1009-1010. Hasegawa, S., Hirano, T., Ogasawara, T., Iwata, M., Bolton, J.P., Akiyama, T. and Arai, S. 1987. Osmoregulatory ability of chum salmon, Oncorhynchus keta, reared in fresh water for prolonged period. Fish Physiol. Biochem. 4:101-110. Helms, L.M.H., Grau, E.G., Shimoda, S.K., Nishioka, R.S. and Bern, H.A. 1987. Studies on the regulation of growth hormone release from the proximal pars distalis of male tilapia, Oreochromis mossambicus, in vitro. Gen. Comp. Endocrinol. 65:48-55. Hirano, T., Ogasawara, T., Bolton, J.P., Collie, N.L., Hasegawa, S. and Iwata, M. 1987. Osmoregulatory role of prolactin in lower vertebrates. In Comparative Physiology of Environmental Adaptations, vol. 1. pp. 112-124. Edited by R. Kirsch and B. Lahlou. Karger, Basal. Kishida, M. and Hirano, T. 1988. Development of radioimmunoassay for eel growth hormone. Nipp. Suis. Gakkaishi 54:1321-1327. Kishida, M., Hirano, T., Kubota, J., Hasegawa, S., Kawauchi, H., Yamaguchi, K. and Shirahata, K. 1987. Isolation of two forms of growth hormone secreted from eel pituitaries in vitro Gen. Comp. Endocrinol. 65:478-488.
165 Komourdjian, M.P., Saunders, R.L. and Fenwick, J.C. 1976. The effect of porcine somatotropin on growth, and survival in seawater of Atlantic salmon (Salmo salar) parr. Can. J. Zool. 54:531-535. Miwa, S. and Inui, Y. 1985. Effects of L-thyroxine and ovine growth hormone on smoltification of amago salmon (Oncorhynchus rhodurus). Gen. Comp. Endocrinol. 58: 436-442. Naito, N., Takahashi, A., Nakai, Y., Kawauchi, H. and Hirano, T. 1983. Immnunocytochemical identification of the prolactin-secreting cells in the teleost pituitary with an antiserum to chum salmon prolactin. Gen. Comp. Endocrinol. 50:282-291. Nishioka, R.S., Richman, N.H., III, Young, G., Prunet, P. and Bern, H.A. 1987. Hypophysectomy of coho salmon (Oncorhynchus kisutch) and survival in fresh water and seawater. Aquaculture 45:167-176. Ogasawara, T., Hirano, T., Akiyama, T., Arai, S. and Tagawa, M. 1989. Changes in plasma prolactin and growth hormone concentrations during freshwater adaptation of juvenile chum salmon (Oncorhynchus keta) reared in seawater for a prolonged period. Fish Physiol. Biochem. (In press). Olivereau, M. 1967. Reactions observes chez l'Anguille maintenue dans un milieu priv6 d'electrolytes, en particulier au
niveau du systeme hypothalamo-hypophysaire. Z. Zellforsch. Mikros. Anat. 80:264-285. Olivereau, M. 1970. Cytologie de l'hypophyse autotransplante chez l'Anguille. Comparison avec celle de Poecilia. Neuroendocrinologie 927:251-260. Olivereau, M. and Ball, J.N. 1970. Pituitary influences on osmoregulation in teleosts. Mem. Soc. Endocrinol. 18:57-85. Olivereau, M., Ollevier, F., Vandesande, F. and Olivereau, J. 1984. Somatostatin in the brain and the pituitary of some teleosts. Immunocytochemical identification and the effect of starvation. Cell Tiss. Res. 238:289-296. Suzuki, R. and Hirano, T. 1987. Prolactin and growth hormone secretion during longterm incubation of the pituitary of chum salmon, Japanese eel and tilapia. Proc. Ist Congr. Asia Oceania Soc. Comp. Endocrinol., pp. 335-336. Suzuki, R., Kishida, M., Ogasawara, T., Hasegawa, S. and Hirano, T. 1987. Prolactin and growth hormone secretion during longterm incubation of the pituitary pars distalis of mature chum salmon, Oncorhynchus keta. Gen. Comp. Endocrinol. 68:76-81. Yamaguchi, K., Yasuda, A., Kishida, M., Hirano, T., Sano, H. and Kawauchi, H. 1987. Primary structure of eel (Anguilla japonica) growth hormone. Gen. Comp. Endocrinol. 66: 447-453.
Growth hormone secretion during longterm incubation of the pituitary of the Japanese eel, Anguilla japonica Reiko Suzuki, Mitsuyo Kishida and Tetsuya Hirano Ocean Research Institute, University of Tokyo, Nakano, Tokyo 164, Japan Keywords: Anguilla japonica, growth hormone, hypothalamic control, osmotic effect, somatostatin
Abstract Growth hormone (GH) secretion from organ-cultured pituitaries of the eel (Anguillajaponica) was studied during incubation in a defined medium for 2 weeks, using a homologous radioimmunoassay which does not distinguish between the two molecular forms of eel GH. The total amount of GH secreted increased gradually during the incubation period; so that the amount of GH released on day 14 was about 30 times greater than that on day 1. On day 14, the proportion of GH released relative to the total amount of GH present (the sum of GH released into the medium and residual content in the pituitary) was 96% and the amount produced on day 14 was 4 times greater than the content in the unincubated pituitary. Somatostatin (SRIF, 1.8 x 10- 7 M) inhibited the increase in GH release. On day 7, the proportion of GH released by pituitaries treated with SRIF (28%) was less than that released by the control pituitary (91 %). There was no significant difference in GH release between the pituitaries incubated in isotonic medium (300 mOsm) and those in hypotonic medium (240 mOsm) for 2 weeks except for the first 3 days, when the pituitaries in hypotonic medium secreted significantly greater amounts of GH than those incubated under isotonic condition. Hypertonic medium (350 mOsm) had no effect on GH release except for significant inhibition on days 6 and 14. When secretion of the two forms of GH (GH I and II) was examined after separation by polyacrylamide gel electrophoresis followed by densitometry, slightly more GH I tended to be secreted than GH II during the culture period, although the effects of SRIF and osmolality of the media on GH I release were similar to those on GH II. It is concluded that GH secretion and production in the eel is mainly under the inhibitory control of hypothalamus, and that osmolality has a minimum influence on the GH release.
Introduction There is increasing evidence suggesting that growth hormone (GH) plays an important role in seawater adaptation of salmonid fishes (Komourdjian et al. 1976; Clarke et al. 1977; Miwa and Inui 1985; Bolton et al. 1987), and an increase in plasma GH levels has been reported in juvenile chum salmon trans-
ferred from fresh water to seawater (Hasegawa et al. 1987; Hirano et al. 1987; Ogasawara et al. 1988). According to Nishioka et al. (1987), hypophysectomized coho salmon are unable to survive in seawater, although they adapt well to fresh water. In tilapia, Helms et al. (1987) reported that hypertonic medium stimulates in vitro release of GH from the pituitary. In contrast, Olivereau and Ball (1970)
Address correspondence to: Reiko Suzuki, Ocean Research Institute, Universily of Tokyo, Nakano, Tokyo 164, Japan.
160 reported a slight reduction in the activity of the GH cells after transfer of the European eel (Anguilla anguilla)from fresh water to seawater, whereas an activation was seen after transfer from fresh water to de-ionized water (Olivereau 1967). GH release from the eel (A. anguilla) pituitary in vitro has been reported to be less in high sodium medium than in low sodium medium (Baker and Ingleton 1975). The hypothalamic control of GH secretion in teleosts has received little attention, although it is suggested to be predominantly under stimulatory control (Ball et al. 1972; Ball 1981). In the eel, GH cells in homotransplanted pituitaries were moderately active histologically (Olivereau 1970). According to Baker and Ingleton (1975), organ-cultured eel pituitaries continued to secrete GH, estimated by densitometry after gel electrophoresis for 2 weeks, indicating autonomy of GH secretion. Thus, hypothalamic control as well as the response to environmental salinity of GH release in the eel would seem to be different from the other teleosts. Recently, two forms of GH have been isolated and fully characterized in the Japanese eel, A. japonica (Kishida et al. 1987; Yamaguchi et al. 1987). A homologous and specific radioimmunoassay (RIA) which does not distinguish between the two forms has also been established recently (Kishida and Hirano 1988). The present study was undertaken to examine the effects of osmolality of the culture media and of somatostatin on GH release, using RIA for measurement of total levels of eel GHs, during incubation in a defined medium for 2 weeks. Secretion of the two GHs was also examined by densitometry after gel electrophoresis.
Materials and methods Cultured immature Japanese eel (Anguilla japonica), weighing 150 to 200 g, were maintained in a freshwater pond. After decapitation, the pituitaries were removed aseptically and preincubated for 1-2h in isotonic medium (Eagle's Minimum Essencial Medium with Earle's salts, 300 mOsm/kg H 20), containing 200 U/ml penicillin, 200 U/ml streptomycin and 0.5 /ag/ml Fungizone (M. A. Bioproducts, Walkersville MD). Two pituitaries were
cultured together for 1-2 weeks at 180C under 95% 02/5070 CO 2 in 0.22 ml medium using a multiwell dish (well diameter 6.4 mm, depth 10 mm). The culture medium contained half the concentrations of the antibiotics used for preincubation. The pH of the medium was adjusted to 7.3-7.4 with sodium bicarbonate. The medium was changed daily. At the end of the culture period, the pituitaries were homogenized in 0.2 ml distilled water. The medium and pituitary extracts were stored at -80°C until hormone assay. The hypotonic medium (240 mOsm) was prepared by diluting the isotonic medium with distilled water, and the hypertonic medium (350 mOsm) by adding mannitol. The effects of medium osmolality were examined using the pituitaries from the same stock of the eel. Somatostatin (SRIF; Peptide Institute, Osaka) was initially dissolved in distilled water and the stock solution was diluted in isotonic medium to 1.8 x 10- 7 M (300 ng/ml). Total contents of the two GHs in the culture medium and the pituitary extract were measured by a homologous RIA using rabbit antiserum against GH I which does not distinguish between two forms of the eel GH (Kishida and Hirano 1988). The culture medium (0.2 ml) and pituitary extract (0.2 ml) were also subjected to polyacrylamide disc electrophoresis at pH 9.5, as described by Davis (1964). Ovine prolactin (NIAMDD-o-PRL-14) was electrophoresed simultaneously as a reference standard. The gel was stained with 0.25% Coomassie brilliant blue. After destaining, the density of protein bands was measured using a densitometer. The bands with Rf 0.29 and 0.36 were identified as GH II and I, respectively (Kishida et al. 1987). The GH contents were expressed as zg ovine PRL equivalent. Immunocytochemical staining of Bouin-fixed, wax-embedded pituitaries was carried out according to the peroxidase-antiperoxidase (PAP) method as described by Naito et al. (1983). Sections of an unincubated pituitary and the pituitary after the culture were stained with the same antiserum as used for RIA. All data were analyzed by Duncan's new multiple range test at the 0.05 level of significance.
161
1
a
2
a
,_
Control
'3' -0t .L c
Z0
En a
0.5
0.5-
la
1
OC
0 a) 0) a
I;
I0)
.
id a, M
.(
I
0 l
1
|
z
3
7
0
ra
days -
1
2 3
6
10
12
14
days
Fig. 1. Secretion of GH from the eel pituitary into hypotonic ( o , 240 mOsm), isotonic ( , 300 mOsm) or hypertonic ( o, 350 mOsm) medium during 14 days, as measured by RIA. GH content in the unincubated pituitary and that remaining after culture are also indicated. Vertical bars indicate standard errors of the means (n = 4-6). * Significantly (p < 0.05) different from the corresponding value in isotonic medium.
Fig. 2. Effect of somatostatin ( , SRIF; 1.8 x 10- 7 M) under isotonic conditions on GH release and on residual GH content in the pituitary, as measured by RIA. Vertical bars indicate standard errors of the means (n = 5-6). * Significantly different (p < 0.05) from the control value in isotonic medium ( A).
7, the relative amount of GH released as a percentage of the total GH (sum of released and residual content) was less in the pituitary treated with SRIF (28%) than in the control pituitary (91 °7).
Results Release of total GH measured by RIA When eel pituitaries were cultured in isotonic medium, GH secretion increased gradually over 2 weeks; the amount of GH release on day 14 was approximately 30 times greater than that on day 1 (Fig. 1). The amount secreted on day 14 was about 4 times greater than the content in the unincubated pituitary and 20 times greater than the residual GH content. The hypotonic medium significantly stimulated GH release for the first 3 days, whereas no effect was seen during the rest of the experiment. Hypertonic medium had no significant effect on GH release except for an inhibition on days 6 and 14. Residual GH contents in the pituitary after culture were significantly less in hypotonic and in hypertonic media than in isotonic medium (Fig. 1). Somatostatin (SRIF, 1.8 x 10- 7 M) inhibited the rise in GH release over 7 days, and the residual GH content of the gland after 7 days was significantly greater than the control level (Fig. 2). On day
Release of two forms of GH separately measured by densitometry The release of both GHs in the isotonic medium increased gradually over 14 days, the increase in GH I secretion being slightly more than GH II (Fig. 3). Altered medium composition had no effect on the release of either form of the hormone except for an inhibition by hypertonic medium on day 14 (Fig. 3). SRIF appeared to inhibit the increase of both GHs but no statistically significant difference was observed using densitometric measurements (Fig. 4). There was no significant difference in the residual hormones in the pituitary after SRIF treatment or altered medium osmolality.
Immunocytochemistry Immunocytochemical examination of the pituitary after culture for 14 days showed that numerous
162 3
a
2
I
2
2
t IFr
-r
a,
M 0, 0, RA
_L i MZ
(3
Cr
0I A v
days
1
3
d days
a -3
3
p2 a'
2 2
I
-3 =-r 1
6
I
I
,
i a
I.
Er N v
g
CT
1
3
6
10
-0
14 days
Fig. 3. Secretion of two forms of GH from the eel pituitary into hypotonic ( o, 240 mOsm), isotonic ( , 300 mOsm) or hypertonic ( o , 350 mOsm) medium during 14 days. GH content in the unincubated pituitary and that remaining after culture are indicated as zg o-PRL equivalent/pituitary. Values were measured by densitometry after gel electrophoresis. Vertical bars indicate standard errors of the means (n = 4-6). * Significantly (p < 0.05) different from the corresponding value in isotonic medium.
immunoreactive GH cells were found in the cultured pituitary, as in the unincubated pituitary (Fig. 5).
Discussion In this study, organ-cultured eel pituitaries secreted gradually increasing amounts of GH over 2 weeks, and the amount of GH present on day 14 (a sum of the amount released into the medium and the residual content in the pituitary) was 4 times greater than the GH content in the unincubated pituitary. Immunocytochemical examination showed that the pituitary after culture for 2 weeks contained as many immunoreactive GH cells as in the unincubated pituitary. Baker and Ingleton (1975) also reported an increase in GH secretion in the European eel
I5
V
n
irao
-o
3 days
Fig. 4. Effect of somatostatin ( , SRIF; 1.8 x 10- 7 M) under isotonic condition on release of GH I and II and on residual content of two forms of GH in the pituitary, as measured by densitometry after gel electrophoresis. Vertical bars indicate standard errors of the means (n = 4-6).
during the first week of culture, although a decline occurred gradually thereafter. These results indicate that the cultured pituitary are viable, and synthesize and secrete an increasing amount of GH during the culture period in a defined medium without supplements such as growth factors, hormones, or fetal calf serum etc. By contrast, the in vitro secretion of GH from the pituitaries of tilapia, rainbow trout and the mature chum salmon decreased gradually during several days of the culture, with considerable amounts of the hormones remaining in the pituitary (Baker and Ingleton 1975; Suzuki and Hirano 1987; Suzuki et al. 1987). Both production and secretion of GH in the eel pituitary seem to be normally under tonic inhibitory control, most likely by the hypothalamus, although presence of potential inhibitory factors in the blood is also possible. This seems to be in agreement with extremely low levels of GH in the eel plasma (less than 0.2 ng/ml; Kishida and Hirano 1988), although this could also be due to an extremely high metabolic clearance of the hormone.
163
Fig. 5. Sagittal section of an eel pituitary cultured for 14 days under isotonic condition (A) and of an unincubated pituitary (B), stained with an anti-eel GH rabbit serum at a dilution of 1:4000. Sections were counterstained with Mayer's hematoxylin. x 60.
In the eel, SRIF fibers terminate on the basal lamina along GH cells, and changes in SRIF immunoreactivity have been observed after starvation suggesting that SRIF regulates GH release in the eel (Olivereau et al. 1984). In the present study, SRIF significantly reduced GH release by day 7, so that residual GH in the pituitary after the culture was greater than in the control pituitary. In contrast, Hall and Chadwick (1978, 1983) reported that hypothalamic extracts stimulated GH release in vitro from the European eel pituitary. In our preliminary study, the eel pituitary incubated with a hypothalamic extract secreted less GH during the 7 day-incubation than the pituitary with an olfactory lobe extract, although the pituitary continued to secrete increasing amounts of GH for 7 days even with the hypothalamic extract (unpublished observation). Thus, GH secretion from the eel pituitary seems to be under both inhibitory and stimulatory control by the hypothalamus, with the inhibitory control being more important.
Secretion of GH from the eel pituitary was not significantly influenced by either hypotonicity or hypertonicity of the culture medium at least over a prolonged period. Recent studies using a perifusion technique revealed that reduced osmotic pressure stimulated PRL release from the tilapia pituitary only for a few hours (Grau et al. 1986, 1987) and from the rainbow trout pituitary for less than 1 hour (Gonnet et al. 1988). Gonnet et al. (1988) suggested that the temporal increase in the hormone secretion by hypotonic medium may be a result of physical modification of the outer cell membrane and not necessarily specific to PRL cells. It is highly probable, therefore, that apparent stimulation of GH release by hypotonic medium for the first 3 days may also be due to mechanical modification. It would be interesting to examine if eel GH has similar osmoregulatory effects in the eel, a catadromous fish, to those seen in the anadromous salmonids, in which GH seems to favor their seawater adaptation (see Bolton et al. 1987; Hirano et al. 1987). The eel GH RIA is more sensitive than the densitometric method; the minimum detectable levels of GH were 10 pg in the RIA (Kishida and Hirano 1988) and 1.5 + 0.3 tAgovine PRL (mean + SEM, n = 6) in the densitometry. Furthermore, interassay coefficients of variation, calculated using the midrange of the assay, were 13.8% (n = 4) in the RIA and 17.7% (n = 3) in the densitometry. These differences in sensitivity and accuracy seem to be a part of the causes for the apparent low degree of correlation between the pattern of GH release measured by RIA and that by the densitometry. For example, a statistically significant decrease in the amount of GH release, as measured by RIA, from the SRIF-treated pituitaries was observed on day 7 (Fig. 2), whereas no significant difference was seen when the densitometry was used to measure the levels of eel GH I and II (Fig. 4). The densitometry seems to be less accurate than the RIA to measure GH amounts in the pituitary, since the pituitary extracts contained proteins with isoelectric points close to those of GH I and II, resulting in increased background staining. Therefore, the residual GH content in the control pituitary as measured by RIA was significantly less than in the SRIF-treated
164 group, whereas no difference was observed by the densitometry (Figs. 2 and 4). Thus, the RIA method seems to provide a 'true' measure of the release of total amount of eel GH. Since the antiserum used for the RIA does not distinguish between the two forms of eel GH, the densitometry following electrophoresis will provide informations on the release of GH I and II separately. Eel GH I has an amino-terminal extension of three amino acid residues, compared with eel GH II, and there is no difference between the growth promoting activities of GH I and II when tested in the juvenile rainbow trout (Kishida et al. 1987; Yamaguchi et al. 1987). In the present study, slightly more GH I tended to be secreted than GH II under the all different culture conditions, although GH I and II were stored in approximately equal quantity both in the unincubated pituitary and in the pituitary after the culture. Further studies are needed to clarify the origin and the physiological role of two forms of eel GH.
Acknowledgements We express our gratitude to Dr. T. Ogasawara and Ms. S. Hasegawa, Ocean Research Institute, University of Tokyo, for their continued interest and encouragement throughout the course of this study, to Professor K. Kohmoto and Dr. S. Sakai, Faculty of Agriculture, University of Tokyo, for their help in the densitometry. We are also grateful to Professor H.A. Bern, University of California, Berkeley, and Dr. B.I. Baker, University of Bath, for their encouragements and suggestions and also for critical readings of the manuscript. This study was aided in part by grants-in-aid for scientific research from the Ministry of Education, and Fisheries Agency.
References cited Baker, B.I. and Ingleton, P.M. 1975. Secretion of prolactin and growth hormone by teleost pituitaries in vitro. II. Effect of salt concentration during long-term organ culture. J. Comp. Physiol. 100:269-282. Ball, J.N. 1981. Hypothalamic control of the pars distalis in fishes, amphibians, and reptiles. Gen. Comp. Endocrinol. 44:135-170.
Ball, J.N., Baker, B.I., Olivereau, M. and Peter, R.E. 1972. Investigations on hypothalamic control of adenohypophysial functions in teleost fishes. Gen. Comp. Endocrinol., Suppl. 3:11-21. Bolton, J.P., Collie, N.L., Kawauchi, H. and Hirano, T. 1987. Osmoregulatory actions of growth hormone in rainbow trout (Salmo gairdneri). J. Endocrinol. 112:63-68. Clarke, W.C., Farmer, S.W. and Hartwell, K.M. 1977. Effect of teleost pituitary growth hormone on growth of Tilapia mossambica and on growth and seawater adaptation of sockeye salmon (Oncorhynchus nerka). Gen. Comp. Endocrinol. 33:174-178. Davis, B.J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, Art. 2:404-427. Gonnet, F., Prunet, P., Tonon, M.C., Dubourg, P., Kah, O. and Vaudry, H. 1988. Effect of osmotic pressure on prolactin release in rainbow trout: in vitro studies. Gen. Comp. Endocrinol. 69:252-261. Grau, E.G., Ford, C.A., Helms, L.M.H., Shimoda, S.K. and Cooke, I.M. 1987. Somatostatin and altered medium osmotic pressure elicit rapid changes in prolactin release from the rostral pars distalis of the tilapia, Oreochromismossambicus, in vitro. Gen. Comp. Endocrinol. 65:12-18. Grau, E.G., Shimoda, S.K., Ford, C.A., Helms, L.M.H., Cooke, I.M. and Pang, P.K.T. 1986. The role of calcium in prolactin release from the pituitary of a teleost fish in vitro. Endocrinology 119:2848-2855. Hall, T.R. and Chadwick, A. 1978. Control of prolactin and growth hormone secretion in the eel Anguilla anguilla. Gen. Comp. Endocrinol. 36:388-395. Hall, T.R. and Chadwick, A. 1983. The effect of thyrotrophin releasing hormone on secretion of prolactin and growth hormone from eel pituitaries incubated in vitro. IRCS Med. Sci. 11:1009-1010. Hasegawa, S., Hirano, T., Ogasawara, T., Iwata, M., Bolton, J.P., Akiyama, T. and Arai, S. 1987. Osmoregulatory ability of chum salmon, Oncorhynchus keta, reared in fresh water for prolonged period. Fish Physiol. Biochem. 4:101-110. Helms, L.M.H., Grau, E.G., Shimoda, S.K., Nishioka, R.S. and Bern, H.A. 1987. Studies on the regulation of growth hormone release from the proximal pars distalis of male tilapia, Oreochromis mossambicus, in vitro. Gen. Comp. Endocrinol. 65:48-55. Hirano, T., Ogasawara, T., Bolton, J.P., Collie, N.L., Hasegawa, S. and Iwata, M. 1987. Osmoregulatory role of prolactin in lower vertebrates. In Comparative Physiology of Environmental Adaptations, vol. 1. pp. 112-124. Edited by R. Kirsch and B. Lahlou. Karger, Basal. Kishida, M. and Hirano, T. 1988. Development of radioimmunoassay for eel growth hormone. Nipp. Suis. Gakkaishi 54:1321-1327. Kishida, M., Hirano, T., Kubota, J., Hasegawa, S., Kawauchi, H., Yamaguchi, K. and Shirahata, K. 1987. Isolation of two forms of growth hormone secreted from eel pituitaries in vitro Gen. Comp. Endocrinol. 65:478-488.
165 Komourdjian, M.P., Saunders, R.L. and Fenwick, J.C. 1976. The effect of porcine somatotropin on growth, and survival in seawater of Atlantic salmon (Salmo salar) parr. Can. J. Zool. 54:531-535. Miwa, S. and Inui, Y. 1985. Effects of L-thyroxine and ovine growth hormone on smoltification of amago salmon (Oncorhynchus rhodurus). Gen. Comp. Endocrinol. 58: 436-442. Naito, N., Takahashi, A., Nakai, Y., Kawauchi, H. and Hirano, T. 1983. Immnunocytochemical identification of the prolactin-secreting cells in the teleost pituitary with an antiserum to chum salmon prolactin. Gen. Comp. Endocrinol. 50:282-291. Nishioka, R.S., Richman, N.H., III, Young, G., Prunet, P. and Bern, H.A. 1987. Hypophysectomy of coho salmon (Oncorhynchus kisutch) and survival in fresh water and seawater. Aquaculture 45:167-176. Ogasawara, T., Hirano, T., Akiyama, T., Arai, S. and Tagawa, M. 1989. Changes in plasma prolactin and growth hormone concentrations during freshwater adaptation of juvenile chum salmon (Oncorhynchus keta) reared in seawater for a prolonged period. Fish Physiol. Biochem. (In press). Olivereau, M. 1967. Reactions observes chez l'Anguille maintenue dans un milieu priv6 d'electrolytes, en particulier au
niveau du systeme hypothalamo-hypophysaire. Z. Zellforsch. Mikros. Anat. 80:264-285. Olivereau, M. 1970. Cytologie de l'hypophyse autotransplante chez l'Anguille. Comparison avec celle de Poecilia. Neuroendocrinologie 927:251-260. Olivereau, M. and Ball, J.N. 1970. Pituitary influences on osmoregulation in teleosts. Mem. Soc. Endocrinol. 18:57-85. Olivereau, M., Ollevier, F., Vandesande, F. and Olivereau, J. 1984. Somatostatin in the brain and the pituitary of some teleosts. Immunocytochemical identification and the effect of starvation. Cell Tiss. Res. 238:289-296. Suzuki, R. and Hirano, T. 1987. Prolactin and growth hormone secretion during longterm incubation of the pituitary of chum salmon, Japanese eel and tilapia. Proc. Ist Congr. Asia Oceania Soc. Comp. Endocrinol., pp. 335-336. Suzuki, R., Kishida, M., Ogasawara, T., Hasegawa, S. and Hirano, T. 1987. Prolactin and growth hormone secretion during longterm incubation of the pituitary pars distalis of mature chum salmon, Oncorhynchus keta. Gen. Comp. Endocrinol. 68:76-81. Yamaguchi, K., Yasuda, A., Kishida, M., Hirano, T., Sano, H. and Kawauchi, H. 1987. Primary structure of eel (Anguilla japonica) growth hormone. Gen. Comp. Endocrinol. 66: 447-453.
