Effects of estrogens in vitro and in vivo on cartilage growth in the tilapia (Oreochromis mossambicus).

To study the effects of estrogens on cartilage growth in the tilapia Oreochromis mossambicus, an epiceratobranchial cartilage radioisotope incorporation assay was employed to measure proteoglycan synthesis and prechondrocyte proliferation by incorporation of radiolabeled sulfate and thymidine, respectively. Cartilage explants were cultured with estrogens with or without recombinant bovine insulin-like growth factor-I (IGF-I). In vitro experiments using the natural teleost estrogen, 17beta-estradiol (E2), showed a trend toward inhibition of sulfate incorporation and an inhibition of thymidine incorporation at higher doses (10 micrograms/ml), but not at physiological levels. E2 also showed a trend toward inhibition of sulfate and thymidine incorporation in the presence of IGF-I. Similar results were found with other estrogenic compounds in vitro: ethinylestradiol, diethylstilbestrol (DES), genistein, and nonylphenol. Ethinylestradiol inhibited sulfate and thymidine incorporation at 1000 ng/ml in the presence of IGF-I. DES inhibited thymidine incorporation at 1000 ng/ml in untreated or IGF-I-exposed cartilage. Genistein inhibited sulfate incorporation at 100 micrograms/ml in IGF-I-exposed cartilage and inhibited thymidine uptake at 1, 10, and 100 micrograms/ml in untreated and IGF-I-exposed cartilage. Nonylphenol inhibited sulfate uptake at 100 microM in untreated and IGF-I-exposed cartilage. Nonylphenol alone at 10 and 100 microM inhibited thymidine uptake. In IGF-I-exposed cartilage nonylphenol inhibited thymidine uptake at 100 microM. Fish receiving estrogen injections (E2 or DES) in vivo at a concentration of 2 micrograms/g body weight showed increased sulfate incorporation by cartilage in vitro. Stimulation in vivo by estrogens, in contrast to the inhibition by high doses in vitro, may be a result of the influence of estrogen on pituitary growth hormone release.

Effects of glucocorticoids on cartilage growth and response to IGF-I in the tilapia (Oreochromis mossambicus).

To study the effects of glucocorticoids and IGF-I on the modulation of growth in the tilapia Oreochromis mossambicus, we employed an epiceratobranchial cartilage radioisotope incorporation assay, wherein radiolabeled sulfate and thymidine uptakes are measured in vitro to indicate proteoglycan synthesis and cell proliferation, respectively. Cartilage explants were cultured with cortisol or dexamethasone with or without recombinant bovine insulin-like growth factor-I. Cortisol directly inhibited sulfate uptake at 100 and 1000 ng/mL concentrations in a concentration-dependent manner but inhibited thymidine uptake significantly only at the 1000 ng/mL concentration. Dexamethasone inhibited sulfate and thymidine uptake at concentrations similar to the effective concentrations of cortisol. Cortisol did not inhibit IGF-I stimulation of sulfate uptake at any of the concentrations tested. Furthermore, cortisol did not inhibit thymidine uptake when IGF-I was present in the medium. Cortisol appears to act directly on cartilage and not by interacting with the IGF-I system. However, the physiologically significant role of cortisol is mainly an inhibitory one on cartilage metabolism. The data generally indicate an inhibitory role for glucocorticoids on cartilage growth but an inability to counter the stimulation of sulfate uptake by IGF-I.

Effects of homologous pituitary hormone treatment on serum insulin-like growth-factor-binding proteins (IGFBPs) in hypophysectomized tilapia, Oreochromis mossambicus, with special reference to a novel 20-kDa IGFBP.

In the circulation, insulin-like growth factors (IGFs) bind to high-affinity-binding proteins. Insulin-like growth-factor-binding proteins (IGFBPs) appear to be present in all vertebrates. To examine the hormonal regulation of serum IGFBPs in a fish, tilapia (Oreochromis mossambicus) were hypophysectomized (Hx) and then treated with homologous tilapia growth hormone (tGH) or either form of tilapia prolactin (tPRL177, tPRL188). Hormones were administered at three doses: 15, 150, and 500 ng/g of body weight. Serum IGFBP profiles were analyzed by SDS-PAGE and Western ligand blotting using 125I-rhIGF-I as a probe. A prominent IGFBP (ca 20 kDa), termed IGFBP-20K, appeared after hypophysectomy. Administration of tGH at all dose levels suppressed this BP and restored levels back to those seen in sham-operated control fish. tPRL177 and tPRL188 were also effective in lowering IGFBP-20K levels. Levels of the 29-kDa IGFBP (termed IGFBP-29K) increased after hypophysectomy; tGH at all doses and tPRL177 at the two lower doses further increased these levels. All doses of tGH, tPRL177, and tPRL188 significantly increased levels of the 32-kDa IGFBP (termed IGFBP-32K). Hypophysectomy significantly lowered levels of the 40-kDa IGFBP (termed IGFBP-40K) below levels seen in the sham-operated controls. tGH treatment significantly raised IGFBP-40K levels at all doses examined, but not to the levels seen in intact tilapia. The 42-kDa IGFBP (termed IGFBP-42K) was not affected by hypophysectomy or hormone replacement. Our data suggest that the novel 20-kDa IGFBP and the 40-kDa IGFBP species may be similar in function to mammalian IGFBP-1 and IGFBP-3, respectively.

Is the primitive regulation of pituitary prolactin (tPRL177 and tPRL188) secretion and gene expression in the euryhaline tilapia (Oreochromis mossambicus) hypothalamic or environmental?

We examined the effects of environmental salinity on circulating levels of the two prolactins (tPRL177 and tPRL188) and levels of pituitary tPRL177 and tPRL188 mRNA in the euryhaline tilapia, Oreochromis mossambicus. Fish were sham-operated or hypophysectomized and the rostral pars distalis (RPD) autotransplanted onto the optic nerve. Following post-operative recovery in (1/4) seawater, tilapia were transferred to fresh water (FW), (1/4) seawater (SW) or SW. Serum tPRL177 and tPRL188 levels in sham-operated and RPD-autotransplanted fish were highest in FW and decreased as salinity was increased. tPRL177 and tPRL188 mRNA levels in RPD implants as well as in pituitaries from the sham-operated fish were also highest in FW and decreased with increasing salinity. Serum osmolality increased with salinity, with the highest levels occurring in the seawater groups. We conclude that some plasma factor (probably plasma osmolality), in the absence of hypothalamic innervation, exerts a direct regulatory action on prolactin release and gene expression in the pituitary of O. mossambicus. This regulation is in accord with the actions of the two prolactins in the freshwater osmoregulation of the tilapia.

Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of the spinal cord.

Urotensin II (UII) is a cyclic peptide initially isolated from the caudal neurosecretory system of teleost fish. Subsequently, UII has been characterized from a frog brain extract, indicating that a gene encoding a UII precursor is also present in the genome of a tetrapod. Here, we report the characterization of the cDNAs encoding frog and human UII precursors and the localization of the corresponding mRNAs. In both frog and human, the UII sequence is located at the C-terminal position of the precursor. Human UII is composed of only 11 amino acid residues, while fish and frog UII possess 12 and 13 amino acid residues, respectively. The cyclic region of UII, which is responsible for the biological activity of the peptide, has been fully conserved from fish to human. Northern blot and dot blot analysis revealed that UII precursor mRNAs are found predominantly in the frog and human spinal cord. In situ hybridization studies showed that the UII precursor gene is actively expressed in motoneurons. The present study demonstrates that UII, which has long been regarded as a peptide exclusively produced by the urophysis of teleost fish, is actually present in the brain of amphibians and mammals. The fact that evolutionary pressure has acted to conserve fully the biologically active sequence of UII suggests that the peptide may exert important physiological functions in humans.

Osmoregulatory actions of growth hormone and prolactin in an advanced teleost.

To date, growth hormone (GH) is known to contribute to seawater adaptation only in salmonid fishes (primitive Euteleostei). Accordingly, the effects of homologous GH and two forms of homologous prolactin (PRL177 and PRL188) on hypoosmoregulatory ability and gill Na+,K(+)-ATPase activity in a more advanced euryhaline cichlid fish, the tilapia (Oreochromis mossambicus), were examined. Following adaptation of hypophysectomized fish to 25% seawater for 3 weeks, fish were given four injections of hormone or vehicle. They were then exposed to 100% seawater for 12 hr and examined for changes in plasma osmolality. Tilapia GH (0.02 and 0.2 microgram/g) significantly improved the ability of tilapia to decrease plasma osmolality following transfer to full-strength seawater, in a dose-related manner. Growth hormone treatment also significantly stimulated gill Na+,K(+)-ATPase activity (0.5 microgram/g). Both tilapia PRLs (PRL177 and PRL188) increased plasma osmolality in 100% seawater and reduced gill Na+,K(+)-ATPase activity, the effects induced by PRL188 being more significant than those by PRL177. Thus, GH may be involved in seawater adaptation of tilapia, a species belonging to the most advanced teleost super-order (Acanthopterygii), whereas both PRLs in tilapia are not involved in seawater adaptation.

Somatotropic actions of the homologous growth hormone and prolactins in the euryhaline teleost, the tilapia, Oreochromis mossambicus.

It is increasingly clear that growth hormone (GH) has growth-promoting effects in fishes, which are mediated in part by the insulin-like growth factor (IGF)-I. Growth-promoting actions of prolactin (PRL) have been reported in higher vertebrates, but are less well established in teleosts. We examined the effects of injecting homologous GH or the two homologous tilapia PRLs (tPRL177 and tPRL188) on the in vitro incorporation of [35S] sulfate (extracellular matrix synthesis) and [3H]thymidine (DNA synthesis) by ceratobranchial cartilage explants and on IGF-I mRNA levels in tilapia liver. Tilapia GH (tGH) and tPRL177 stimulated sulfate uptake at the highest doses examined. Thymidine incorporation was stimulated by tPRL177. tPRL188 was without these effects. Consistent with its somatotropic actions, tGH elevated IGF-I mRNA levels in the liver. tPRL177 also elevated liver IGF-I levels. Consistent with the previously described osmoregulatory actions of GH and PRL in teleosts, we observed that tGH elevated and tPRL177 and tPRL188 lowered levels of gill Na+,K+-ATPase activity. High-affinity, low-capacity binding sites for tGH in the tilapia liver were identified. tPRL177 binds with lower affinity than tGH to these sites but can displace 125I-labeled tGH from its receptor. The ability of tPRL177 to displace tGH was similar to that of ovine GH. tPRL188 did not displace 125I-labeled tGH binding. Collectively, this work suggests that tPRL177 may possess somatotropic actions similar to tGH, but only in freshwater tilapia where tPRL177 levels are sufficiently high for it to act as a competitive ligand for GH receptors.

Mouse anococcygeus muscle: sexual dimorphism and responsiveness to sex hormones.

The anococcygeus muscle (AcM) is one of a pair of thin sheets of smooth muscle inserting on the rectum, having a tendinous origin largely on sacral vertebrae. The cross-sectional area of AcM in the juxtarectal region in 90-day-old male mice was significantly larger than that in females of three strains: BALB/cCrgl, ICR/Jcl and C57BL/Tw. The AcM area in female mice showed strain differences: BALB/c > ICR > C57BL. Five daily injections of testosterone into newborn ICR mice from the day of birth significantly increased the areas of AcM in both sexes at 30 days of age, but five daily injections of oestradiol-17 beta (OE) decreased them. The AcM area in 60-day-old ICR male mice castrated at 30 days of age was significantly smaller than in intact males, and that in ovariectomized females was significantly larger than in intact females. In both sexes, implantation of a testosterone pellet (12 mg) into gonadectomized mice on the day of gonadectomy stimulated the growth of AcM, and implantation of an OE pellet (12 mg) inhibited the growth of AcM. The AcM in both ICR and C57BL strains showed positive androgen receptor and oestrogen receptor immunostaining at 15 days. Female ICR mice exposed neonatally to diethylstilboestrol (DES) had significantly larger AcM than controls; ovariectomy at 30 days of age did not change the AcM area in 60-day-old DES-exposed mice. However, male mice exposed neonatally to DES had significantly smaller AcM than controls; castration at 30 days of age nullified this inhibition. These results suggest that both androgen and oestrogen play an important role in sexual dimorphism of the mouse AcM. Neonatal exposure to DES (but not to oestradiol) had an irreversible stimulatory effect on the AcM area in female mice.

A low-molecular-weight (25-kDa) IGF-binding protein is increased with growth inhibition in the fasting striped bass, Morone saxatilis.

The effect of fasting on circulating IGFBPs in the striped bass was assessed in relation to changes in growth and metabolism. Thirty-day-fasted (30DF) and 60-day-fasted (60DF) fish, and 60DF fish refed for 14 additional days (REFED), were compared with control, fed fish. Growth and metabolic status of each animal were assessed by determining body length (BL) and body weight (BW) changes, hepatosomatic index (HSI), condition factor (CF), and serum glucose concentration, and by assaying for incorporation of [35S]sulfate (proteoglycan synthetic activity) and [3H]thymidine (mitotic activity) in ceratobranchial cartilage explants in vitro. Serum IGFBP concentrations were assessed by a Western ligand blot procedure using 125I-labeled human IGF-I tracer. Both 30DF and 60DF fish exhibited hypoglycemia and reduced HSI and CF, and their BL and BW growth rates were significantly inhibited. Strongly correlated with the inhibited body growth indices were significantly depressed levels of cartilage [35S]sulfate incorporation in both 30DF and 60DF animals. The 60DF group also exhibited reduced [3H]thymidine incorporation. Associated with this growth inhibition was a dramatic increase in the serum levels of a 25-kDa IGFBP (sbIGFBP-1). A 35-kDa IGFBP (sbIGFBP-3), on the other hand, was not significantly altered with fasting. All fasting-induced changes in growth, metabolism, and IGFBP levels were restored in the REFED group. These results demonstrate that an IGFBP of low molecular weight is increased with growth inhibition in the fasting striped bass, suggesting that a teleost fish counterpart to mammalian IGFBP-1 may exist.

Effects of sex hormones on oncogene expression in the vagina and on development of sexual dimorphism of the pelvis and anococcygeus muscle in the mouse.

Neonatal treatment of female mice with diethystilbestrol (DES) is known to induce ovary-independent persistent proliferation and cornification of vaginal epithelium. This irreversibly changed vaginal epithelium persistently expressed higher levels of c-jun and c-fos mRNAs, which was not altered by postpubertal estrogen. Sexual dimorphism was encountered in mouse pelvis and anococcygeus muscle. Postpubertal estrogen changed the shape of the pelvis to the female type and postpubertal androgen changed it to the male type. Neonatal exposure to DES and to the antiestrogen tamoxifen altered the developmental pattern of the pelvis, which contained lower concentrations of calcium and phosphorus than controls. The size of anococcygeus muscle was increased by postpubertal androgen but decreased by postpubertal estrogen. However, neonatal estrogen (DES) exposure permanently enlarged the anococcygeus muscle. Thus, neonatal treatment of mice with estrogen and antiestrogen results in irreversible changes in nonreproductive as well as reproductive structures.

In vitro secretion of insulin-like growth factor-binding proteins from liver of striped bass, Morone saxatilis.

In vitro secretion of insulin-like growth factor-binding proteins (IGFBPs) from liver of striped bass (sb: Morone saxatilis) was studied using a simple organ-culture system. Liver cubes (1 mm3) were cultured in minimum essential medium with Earle's salts containing 0.1% bovine serum albumin and 100 U/ml penicillin in 5% CO2/95% O2 at 16 degrees. The amount of double-stranded DNA in these cultured liver cubes did not change by 192 hr in the culture, but decreased by 216 hr. Four IGFBPs (a 23- to 24-kDa protein, a 28- to 30-kDa protein, a 35- to 39-kDa protein, and an 85- to 90-kDa protein) were identified in striped bass serum by Western ligand blotting; two of these IGFBPs, 23-24 kDa (sbIGFBP-1) and 28-30 kDa (sbIGFBP-2), were consistently detected in culture media by Western ligand blot analysis. The intensity of the blot for sbIGFBP-2 was consistently greater than that of sbIGFBP-1, which was no longer secreted after 96 hr in culture. The effects of hormones and growth factors on IGFBP secretion by liver tissue were measured after 48 hr in culture. sbIGFBP-1 in the medium was significantly decreased by adding ovine prolactin (10 micrograms/ml), bovine insulin (100 micrograms/ml), and bovine IGF-I (100 ng/ml), but was increased by 17 beta-estradiol (E2: 5 and 50 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of environmental salinity on pituitary growth hormone content and cell activity in the euryhaline tilapia, Oreochromis mossambicus.

Studies were undertaken to determine whether several indicators of growth hormone (GH) cell activity, namely GH content, fine structure, and volume of the GH region, differ in the pituitaries of freshwater (FW) and seawater (SW) tilapia, Oreochromis mossambicus. Tilapia raised from the stage of yolk-sac absorption for 7 months in SW contain significantly more GH in their pituitaries than in those of fish reared in FW. Pituitary growth hormone content in tilapia raised in FW for 7 months and transferred to SW for 49 days is greater than that in sibling tilapia retained in FW. Conversely, GH content is significantly lower in the pituitaries of SW-reared tilapia transferred to FW for 49 days than that in the pituitaries from fish retained in SW. Likewise, the volume of the GH region and activity of the GH cells are enhanced in pituitaries from SW-reared tilapia over that seen in pituitaries from FW fish. Taken together, all data indicate heightened GH cell activity in SW-raised tilapia and suggest that GH may play a causal role in the greater growth rates observed in SW tilapia compared to FW fish and/or that GH may be involved in SW osmoregulation. The latter suggestion is supported, in part, by our observation that in vivo oGH treatment (2 micrograms/g body wt) stimulated gill Na+,K(+)-ATPase activity.

In vitro study of the effect of urotensin II on corticosteroid secretion in the frog Rana ridibunda.

Urotensin II is a cyclic dodecapeptide that was originally isolated from the fish urophysis, the terminus of a neurosecretory system located in the caudal area of the spinal cord. We have recently isolated and characterized urotensin II in the brain of a tetrapod, the frog Rana ridibunda. Recent reports, suggesting that urotensin II may stimulate cortisol secretion in fish, prompted us to investigate the possible effects of fish and frog urotensin II on corticosteroid secretion in amphibians. Exposure of perifused frog adrenal slices to goby (Gillichthys mirabilis) urophysis extracts induced a marked stimulation of corticosterone and aldosterone secretion. In contrast, at concentrations ranging from 10(-10) to 10(-6) M, synthetic goby urotensin II had no effect on corticosteroid production. Similarly, infusion of synthetic frog urotensin II (10(-10) to 10(-6) M) did not modify the spontaneous release of corticosterone and aldosterone. In addition, frog urotensin II had no effect on ACTH- and angiotensin II-induced secretion of corticosteroids. These results show that in frog, urotensin II does not modulate spontaneous and ACTH- or angiotensin II-evoked adrenal steroidogenesis.

Physiology of seawater acclimation in the striped bass, Morone saxatilis (Walbaum).

Several experiments were performed to investigate the physiology of seawater acclimation in the striped bass, Morone saxatilis. Transfer of fish from fresh water (FW) to seawater (SW; 31-32 ppt) induced only a minimal disturbance of osmotic homeostasis. Ambient salinity did not affect plasma thyroxine, but plasma cortisol remained elevated for 24h after SW transfer. Gill and opercular membrane chloride cell density and Na(+),K(+)-ATPase activity were relatively high and unaffected by salinity. Average chloride cell size, however, was slightly increased (16%) in SW-acclimated fish. Gill succinate dehydrogenase activity was higher in SW-acclimated fish than in FW fish. Kidney Na(+), K(+)-ATPase activity was slightly lower (16%) in SW fish than in FW fish. Posterior intestinal Na(+),K(+)-ATPase activity and water transport capacity (Jv) did not change upon SW transfer, whereas middle intestinal Na(+),K(+)-ATPase activity increased 35% after transfer and was correlated with an increase in Jv (110%). As salinity induced only minor changes in the osmoregulatory organs examined, it is proposed that the intrinsic euryhalinity of the striped bass may be related to a high degree of "preparedness" for hypoosmoregulation that is uncommon among teleosts studied to data.

Effects of prolactins on the chromatophores of the tilapia, Oreochromis niloticus.

Using isolated scales and split-fin preparations of the tilapia Oreochromis niloticus, the effects of a pair of prolactins of the tilapia Oreochromis mossambicus (tPRL177 and tPRL188) and of ovine prolactin (oPRL) on chromatophores were studied in vitro. These peptides caused melanosome aggregation and dispersion of xanthosomes, especially in the split preparations. Their relative effectiveness was as follows: tPRL177 > oPRL > tPRL188. Moreover, tPRL177 at 100 nM induced a high level of pigment dispersion in cultured xanthophores and erythrophores, but tPRL188 at the same concentration did not have this effect. We also examined the responses of chromatophores to oPRL in primary cell culture and found that xanthophores and erythrophores respond to the peptide by pigment dispersion in a dose-dependent manner, whereas cultured melanophores showed little aggregation of pigment. In denervated melanophores in the split-fin preparations, tPRL177 failed to induce aggregation of pigment. From these results, it was concluded that prolactin affects brightly pigmented cells of the tilapia directly, but affects melanophores indirectly. Norepinephrine which might leak from varicosities of chromatic nerve fibers by virtue of the action of prolactin molecules may be responsible for melanosome aggregation.

Epidermal growth factor receptor levels in reproductive organs of female mice exposed neonatally to diethylstilbestrol.

Binding of epidermal growth factor (EGF) to membrane preparations of vagina, uterus, ovary, oviduct, and liver was examined in mice treated neonatally with diethylstilbestrol (DES) and compared with that in untreated mice. Binding in the vagina (12.5 +/- 0.73 fmol/mg protein) was somewhat higher than in the uterus (8.0 +/- 0.34 fmol/mg protein). Level of specific binding was of the order: liver (18.4 +/- 1.09 and 16.0 +/- 1.53 fmol/mg protein) > vagina (12.5 +/- 0.73 and 8.2 +/- 0.57 fmol/mg protein) > uterus (8.0 +/- 0.34 and 6.8 +/- 0.56 fmol/mg protein) > ovary (6.8 +/- 0.36 and 8.0 +/- 1.05 fmol/mg protein) > oviduct (2.1 +/- 0.32 and 1.7 +/- 0.05 fmol/mg protein) in control and neonatally DES-exposed mice, respectively. Thus, neonatal DES exposure significantly lowered the binding site level only in the vagina, without modifying the binding affinity (Kd = 5.4 x 10(-9) M in controls vs 4.6 x 10(-9) M in DES-exposed mice). Reduction of EGF receptor level in the vagina correlates with ovary-independent persistent proliferation and keratinization of the vagina induced by neonatal DES exposure. EGF receptors were immunohistochemically demonstrated in epithelial cells of vagina, uterus, and oviduct and in stromal cells in uterus and oviduct using a polyclonal antibody to human EGF receptor protein.

In-vitro effects of insulin-like growth factor-I on gill Na+,K(+)-ATPase in coho salmon, Oncorhynchus kisutch.

The effect of ovine GH (oGH) in vivo and recombinant bovine insulin-like growth factor-I (rbIGF-I) in vitro on gill Na+,K(+)-ATPase activity was investigated in two seasonal experiments conducted during the parr-smolt transformation period of coho salmon. In 1991, when fish were held under a photoperiod of 12 h light : 12 h darkness, the stimulatory effect of oGH (1 microgram/g) on gill Na+,K(+)-ATPase in vivo decreased at the time of expected parr-smolt transformation. Gill Na+,K(+)-ATPase from control fish was insensitive to rbIGF-I in vitro from February to June, whereas GH treatment induced sensitivity to rbIGF-I (100-1000 micrograms/l) in vitro in February and March, but not later in development. In 1992, when fish were held under natural conditions, oGH (4 micrograms/g) stimulated gill Na+,K(+)-ATPase in vivo from February to July. There was, however, of pronounced developmental change in sensitivity of gill Na+,K(+)-ATPase to rbIGF-I in vitro. In February, gills from control fish were insensitive, but oGH treatment in vivo induced sensitivity to rbIGF-I in vitro (100-1000 micrograms/l). In April and May, control fish were sensitive to rbIGF-I in vitro. This sensitivity was not further potentiated by oGH treatment in vivo. In June, gills from control or oGH-treated fish were not sensitive to rbIGF-I in vitro, but in July exogenous oGH again induced gill tissue sensitivity to rbIGF-I at 1000 micrograms/l. Both studies showed that rbIGF-I stimulates gill Na(+),K(+)-ATPase directly; an ability that may depend on priming by endogenous or exogenous GH. This supports the role of IGF-I as an endocrine mediator for GH action during parr-smolt transformation.

Experimental diabetes mellitus in a teleost fish. II. Roles of insulin, growth hormone (GH), insulin-like growth factor-I, and hepatic GH receptors in diabetic growth inhibition in the goby, Gillichthys mirabilis.

Insulin-dependent diabetes mellitus (IDDM), when untreated or poorly controlled in mammals, results in growth retardation. To determine whether the same relationship exists in an ectothermic vertebrate, IDDM-like symptoms were induced in a teleost fish, the goby Gillichthys mirabilis, by surgical removal of its pancreatic endocrine (islet) organ. Isletectomized (Ix) gobies lost body weight, their skeletal growth was retarded, as measured by changes in body length, and they exhibited a 50% reduction in cartilage 35SO4 incorporation in vitro, consistent with changes that occur in mammals with IDDM. Injections of bovine insulin into the Ix fish restored body growth parameters to control levels and stimulated cartilage 35SO4 incorporation in a dose-related manner. In contrast to mammals with IDDM, which are resistant to GH action, injection of teleost GH stimulated cartilage 35SO4 incorporation in the Ix fish. Furthermore, whereas cartilage from rats with IDDM is resistant to stimulation by insulin-like growth factor-I (IGF-I) in vitro, cartilage explants from the Ix fish were highly responsive to recombinant bovine IGF-I, exhibiting a dose-dependent stimulation of 35SO4 incorporation. As far as we are aware, these results represent the first demonstration of diabetic growth inhibition in an ectothermic vertebrate. This inhibition is similar to that which occurs in mammals with IDDM in some respects, but is different in others, as the diabetic fish did not develop resistance to growth stimulation by either GH or IGF-I. While these results support a role for insulin in maintaining the GH-IGF-I-growth axis in this ectothermic vertebrate, there may be important differences in the role of insulin in the promotion of anabolic processes.

Effects of salinity on chloride cells and Na+, K(+)-ATPase activity in the teleost Gillichthys mirabilis.

1. Longjawed mudsuckers, Gillichthys mirabilis, in 30 ppt seawater (SW) were transferred to 1.5, 30 and 60 ppt SW. 2. In the first 1-3 days after transfer, plasma chloride level and plasma osmolarity rose in the 60 ppt SW fish, and decreased in the 1.5 ppt SW fish. 3. By day 21, however, plasma chloride and osmolarity were at or near the levels seen in the controls (30 ppt). 4. Branchial and jawskin Na+, K(+)-ATPase activities were high in all salinities, and did not differ significantly among treatments. 5. The vital fluorescent stains DASPEI and anthroylouabain were used to detect mitochondria and Na+, K(+)-ATPase, respectively, in chloride cells. 6. Both stains indicated that jawskin chloride cell density did not differ among treatment groups. 7. In contrast, chloride cell size increased significantly with increasing salinity. 8. The chloride cells of fish in 60 ppt SW were noticeably angular in outline, whereas those of both the 1.5 and 30 ppt SW fish were circular. 9. The results are discussed in relation to the ion transport requirements encountered in the intertidal habitat of the mudsucker.

Effects of acclimation to hypertonic environment on plasma and pituitary levels of two prolactins and growth hormone in two species of tilapia, Oreochromis mossambicus and Oreochromis niloticus.

Specific radioimmunoassays (RIAs) for the pair of tilapia prolactins (tPRLs) and growth hormone (tGH) were developed using antisera raised in rabbits. Anti-tPRL177 did not cross-react with tPRL188 and tGH. Anti-tPRL188 did not cross-react with tPRL177 and showed slight cross-reaction (3.1%) with tGH. Anti-tGH showed negligible cross-reactions with tPRL177 (0.4%) and tPRL188 (1.6%). Pituitary homogenates and plasma from Oreochromis niloticus exhibited displacement curves parallel to the standards in the three RIAs. Plasma from hypophysectomized O. niloticus showed no cross-reaction in any of the three RIAs. Plasma and pituitary levels of the two PRLs in O. mossambicus in freshwater did not differ significantly from each other, whereas in O. niloticus, the levels of PRL177 were significantly greater than those of PRL188 in both plasma and pituitary. After acclimation for 3-4 weeks in seawater (O. mossambicus) or 50% seawater (O. niloticus), the levels of both PRLs decreased significantly compared to their levels in freshwater. Acclimation to a hypertonic environment did not affect plasma and pituitary GH levels in either species. Immunocytochemical staining of the pituitary of O. niloticus revealed colocalization of both PRLs in rostral pars distalis. Our findings suggest that the synthesis and secretion of the two tPRLs could be independently regulated in the same cells.