Tetsuya Hirano

DEVELOPMENT OF THE PITUITARY, THYROID AND INTERRENAL GLANDS AND APPLICATIONS OF ENDOCRINOLOGY TO THE IMPROVED REARING OF MARINE FISH LARVAE

Abstract A key to success in the culture of marine fish is the mass production of high quality fry, a process largely dependent on successful first feeding and normal development and growth of fish larvae. In this regard it is important to examine the structural and functional development of the endocrine system during early ontogeny of marine fish. To characterize early endocrine development a comparative study was conducted with various marine fish. The age when the pituitary gland differentiated, assessed by immunohistochemical detection of GH and PRL, varied among species, but generally coincided with the time of eye pigmentation, when yolk absorption is very rapid in most marine fish larvae. Thyroid follicles and pancreatic islets first appeared at about the same time, while interrenal cells appeared later. Tissue concentrations of thyroid hormones decreased during embryonic development to nearly undetectable levels at the completion of yolk absorption. During early larval life thyroid hormone levels increased gradually and were significantly elevated during the transformation to the juvenile stage, especially in flatfish. Tissue concentrations of cortisol showed nearly the same developmental pattern, but increased prior to thyroid hormone levels. GH and PRL exhibited different developmental patterns. GH increased continuously during larval growth while PRL exhibited nearly the same pattern as cortisol in Japanese flounder. Based on these findings it is concluded that the endocrine system becomes functional before the completion of yolk absorption in most marine fish, when the switch from a maternal (yolk) to an endogenous larval hormone source occurs. A preliminary experiment on T 4 supplementation indicated that intact protein ingestion by pinocytosis in the rectal epithelium and fat absorption in the intestinal epithelium were enhanced. Potential benefits of exogenous hormone treatment are discussed particularly in relation to digestive functions.

Kinetic studies of growth hormone and prolactin during adaptation of coho salmon, Oncorhynchus kisutch, to different salinities.

The kinetics of growth hormone (GH) and prolactin (PRL) in coho salmon (Oncorhynchus kisutch) transferred from fresh water (FW) to seawater (SW) and vice versa were examined to help clarify the osmoregulatory roles of the two hormones during periods of migration to different salinities. Chum salmon GH or PRL was administered by a single injection intraarterially, and metabolic clearance rate (MCR) and secretion rate (SR) of injected hormones were calculated from the disappearance of the hormones from the plasma. When coho salmon smolts were acclimated to SW, MCR, SR, and plasma level of GH in SW-adapted (2-3 weeks) fish were twice as great as those in fish in FW. On the other hand, there was no difference in the kinetics of GH between the adult coho salmon in SW and those adapted to FW (2-3 weeks). The transfer of the adult coho salmon from SW to FW was followed after 2 days by a rise in plasma level and SR of PRL, which tended to stay at high levels after 2-3 weeks. The MCR of PRL increased significantly after 2-3 weeks in FW. These results support the likelihood of an important role of GH in SW adaptation and of PRL in FW adaptation in coho salmon.

Hepatic receptors for homologous growth hormone in the eel.

The specific binding of 125I-labeled eel growth hormone (eGH) to liver membranes of the eel was examined. The specific binding to the 10,000g pellet was greater than that to the 600g pellet. The specific binding was linear up to about 100 mg fresh tissue, and was saturable with increasing amounts of membrane. The specific binding was pH-, temperature-, and time-dependent, with the optimum pH at 7.4, and greater specific binding was obtained at 15 and 25 degrees than at 35 degrees. Scatchard analysis of liver binding gave an association constant of 1.1 x 10(9) M-1 and a capacity of 105 fmol/mg protein. The receptor preparation was highly specific for GHs. Natural and recombinant eel GHs as well as recombinant salmon GH competed equally with 125I-eGH for the receptor sites of the 10,000g liver membrane. Ovine GH was more potent in displacing the labeled eGH than the homologous eel hormone. Tilapia GH and ovine prolactin (PRL) were needed in greater amounts (40 times) than eGH to displace the labeled eGH. Salmon and tilapia PRLs were still less potent (500 times) than eGH. There was no displacement with eel PRL. No significant change in the specific binding was seen 1 week after hypophysectomy, whereas injection of eGH into the hypophysectomized eel caused a significant reduction after 24 hr. The binding to the membrane fractions from gills, kidney, muscle, intestine, and brain was low and exclusively nonspecific, indicating the presence of specific GH receptors predominantly in the liver.

Changes in plasma hormone levels during loss of hypoosmoregulatory capacity in mature chum salmon (Oncorhynchus keta) kept in seawater

Returning chum salmon (Oncorhynchus keta) in northern Honshu Island, Japan, complete gonadal maturation while in the bay. Mature fish caught in the bay failed to survive in seawater for more than a week, whereas they adapted to fresh water efficiently. Mortality in seawater seems to be due primarily to an increased plasma osmolality. Maladaptation to seawater was more pronounced in the fish caught deep in the bay than those caught outside the bay, and also greater in females than in males. In close correlation with the increased plasma osmolality and electrolyte concentrations, plasma levels of cortisol and growth hormone increased in the fish kept in seawater. Cortisol and growth hormone may be secreted in response to the increased plasma osmolality and would not be the direct cause of the maladaptation to seawater. Plasma prolactin remained low in the seawater fish, indicating that the increased secretion of prolactin, a freshwater-adapting hormone, is not the cause of maladaptation to seawater either. On the other hand, when the fish caught in the river were kept in fresh water, an increase in plasma prolactin concentrations was seen, particularly in females, whereas no significant change was seen in plasma cortisol and growth hormone. Concentrations of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one in the female and of testosterone in both the male and female were extremely high in the bay fish and decreased slightly but significantly after 7 days in fresh water. The gonadal steroids may have inhibitory effects on osmoregulation in the mature salmon in seawater.

Hypoosmoregulatory ability of eyed-stage embryos of chum salmon

To evaluate the osmoregulatory ability of eyedstage embryos of laboratory-reared chum salmon, Oncorhynchus keta, we examined changes in osmolality of the perivitelline fluid and blood following transfer to 50 and 100% seawater (SW), together with morphological changes in chloride cells present in the yolk sac membrane. Transfer to SW did not cause any significant change in the whole egg weight. However, the embryos shrank when the eggs were transferred to SW, whereas the perivitelline space increased at the expense of the embryo. Osmolality of the perivitelline fluid increased rapidly to reach environmental levels 3 h after transfer, indicating that the egg shell is permeable to ions and water. Blood osmolality increased after transfer to SW, reached a peak level at 3 h, and then decreased gradually. The chloride cells in the yolk sac membrane became activated following transfer, as shown by increased cell size and frequent appearance of apical openings. These results indicate that the eyed-stage embryos of chum salmon possess hypoosmoregulatory ability and that chloride cells in the yolk sac membrane may be involved in salt extrusion, in place of gill chloride cells, during the late embryonic stage.

Inhibition of growth hormone synthesis by somatostatin in cultured pituitary of rainbow trout

SummaryWhen the pituitary of rainbow trout (Oncorhynchus mykiss) was incubated in a serum-free medium, a high level of growth hormone release as well as an activation of growth hormone synthesis were observed, suggesting the existence of hypothalamic inhibitory factor(s) on growth hormone synthesis. Although an inhibitory effect of somatostatin on growth hormone release is well established in both mammals and teleosts, an effect on growth hormone synthesis has not been demonstrated. In this study, we examined the effect of somatostatin on growth hormone synthesis in organ-cultured trout pituitary using immunoprecipitation and Northern blot analysis. Somatostatin inhibited growth hormone release from the cultured pituitary within 10 min after addition without affecting prolactin release. Incubation of the pituitary with somatostatin also caused a significant reduction in newly-synthesized growth hormone in a dose-related manner, as assessed by incorporation of [3H]leucine into immunoprecipitable growth hormone. There were no changes in the level or molecular length of growth hormone mRNA after somatostatin treatment, as assessed by Northern slot blot and Northern gel blot analyses. Human growth hormone-releasing factor stimulated growth hormone release, although the spontaneous synthesis of growth hormone was not augmented. However, somatostatin-inhibited growth hormone synthesis was restored by growth hormone-releasing factor to the control level. The spontaneous increase in growth hormone synthesis observed in the organ-cultured trout pituitary may be caused, at least in part, by the removal of the inhibitory effect of hypothalamic somatostatin.

Thyroid hormone concentrations in the gonads of wild chum salmon during maturation

Changes in gonadal and plasma concentrations of thyroid hormones were examined at various stages of maturation in chum salmon (Oncorhynchus keta) caught in the Bering Sea and the Bay of Alaska. Plasma concentrations of thyroxine (T4) were less than 5 ng ml−1, and those of 3,5,3′-triiodo-L-thyroxine (T3) were less than 2 ng ml−1 I in both males and females, regardless of the degree of sexual maturity or the gonadosomatic index (GSI).There was no clear relationships between circulating thyroid hormone levels and tissue levels. The ovarian T4 concentrations were undetectable (less than 0.2 ng g−1) or less than 2 ng g−1 when GSI was lower than 1%, but increased thereafter and reached a plateau of 8–10 ng g−1 when GSI became 2%. The ovarian T3 concentrations were about 5 ng g−1 when GSI was 1%, increased to a maximum level (20 ng g−1) when GSI was about 2%, and decreased to a constant level of 10 ng g−1 thereafter. The T4 and T3 content in single oocyte increased proportionally to the oocyte volume, indicating a constant incorporation of the hormones into the oocyte.The T4 concentrations in the testis were 1 ng g−1 or less regardless of the GS1. On the other hand, the T3 concentrations were highest (15 ng g−1) when the GSI was less than 1%, decreased thereafter when spermatocytes appeared in the testis, and became about 5 ng g−1 I in testes containing spermatozoa, raising the possibility of a role for T3 during early gamete and/or gonad maturation of testes.

Effects of osmotic pressure on prolactin and growth hormone secretion from organ-cultured eel pituitary

SummaryEffects of medium osmotic pressure on the release of prolactin (PRL) and growth hormone (GH) from the pituitary of the Japanese eel, Anguilla japonica, were examined during long-term organ culture in a defined medium. Prolactin and GH release, as measured by homologous radioimmunoassays, increased gradually for 7 days during incubation in isosmotic medium (295 mOsmolal). On day 7, 3 to 5 times more PRL and GH were released than on day 1. The amount of GH released was about 100 times greater than that of PRL. Electron microscopic observation revealed that both PRL and GH cells were in good condition after 7 days incubation. The reduction of medium osmotic pressure from 295 (isosmotic) to 235 or 260 mOsmolal significantly stimulated PRL release for 4 days. By contrast, an increase in medium osmolality from 295 to 360 mOsmolal was without effect. These treatments produced no significant alterations in GH release. The stimulatory effect of hyposmotic medium (235 mOsmolal) was no longer evident by 12 h after the pituitaries were returned to isosmotic medium. The isosmotic but low-sodium medium, prepared by adding mannitol to the hyposmotic medium, did not stimulate PRL release from the pituitary. These results indicate that plasma osmolality may be an important physiological factor controlling PRL release during freshwater adaptation of the eel.

Development of a homologous radioimmunoassay for eel prolactin.

A highly specific and homologous radioimmunoassay (RIA) for the measurement of prolactin (PRL) in the plasma and the pituitary of the eel was developed using a rabbit antiserum to eel PRL. PRL was purified from the pituitary of Japanese eel (Anguilla japonica). Pituitary extracts and plasma from the Japanese and European eels exhibited displacement curves parallel to the eel PRL standard. Plasma and pituitary extracts from chum salmon, rainbow trout, Japanese charr, tilapia, goldfish, and carp, as well as plasma from hypophysectomized eel, showed negligible cross-reactivity. PRL and growth hormone (GH) preparations from chum salmon, tilapia, and sheep, carp PRL, and eel GH did not cross-react with the antibody. The RIA sensitivity was less than 0.1 ng eel PRL per milliliter. Intra- and interassay coefficients of variations were 2.4 and 11.8%, respectively. The immunoreactive PRL levels in plasma and pituitary of the eel adapted to 50% seawater were significantly lower than those of the eel in fresh water. Plasma PRL levels increased maximally 2 days after transfer from seawater to fresh water, as would be expected from the well-established role of PRL in freshwater adaptation in several euryhaline teleosts.