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The insulin-like growth factor (IGF) system plays a central role in the neuroendocrine regulation of growth in all vertebrates. Evidence from studies in a variety of vertebrate species suggest that this growth factor complex, composed of ligands, receptors, and high-affinity binding proteins, evolved early during vertebrate evolution. Among nonmammalian vertebrates, IGF signaling has been studied most extensively in fish, particularly teleosts of commercial importance. The unique life history characteristics associated with their primarily aquatic existence has fortuitously led to the identification of novel functions of the IGF system that are not evident from studies in mammals and other tetrapod vertebrates. Furthermore, the emergence of the zebrafish as a preferred model for development genetics has spawned progress in determining the requirements for IGF signaling during vertebrate embryonic development. This review is intended as a summary of our understanding of IGF signaling, as revealed through research into the expression, function, and evolution of IGF ligands, receptors, and binding proteins in fish.

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., (Copyright 2001 Academic Press.)

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., (Copyright 2001 Academic Press.)

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.

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.

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.

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.

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.

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.

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.

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 (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)

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.

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.

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.

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.

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.

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.

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.

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.

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.

The relationship between plasma thyroid hormone concentrations and the thyroid hormone concentrations in selected tissues was examined throughout the spring during the typical course of parr-smolt transformation in coho salmon (Oncorhynchus kisutch) in fresh water and also in coho salmon moved prematurely to seawater. The thyroid hormones thyroxine (T4) and triiodothyronine (T3) were extracted from brain, liver, and muscle tissue. The T4 and T3 concentrations in the extracts and plasma were measured by radioimmunoassay. The peak in plasma T4 occurred in late April; however, the concentration of T4 in the brain and liver increased before levels of T4 in plasma increased. During the rise in plasma T4, the T4 content in muscle decreased. Plasma T3 concentrations were unchanged in March and April, but decreased in May. Transfer to seawater eliminated the late April peak in plasma T4 levels, indicating suppressed thyroid activity; however, the tissues of salmon in seawater contained more T3 than tissues of salmon in fresh water at this time. These findings indicate complex peripheral regulation of thyroidal status in this teleost and represent the first bridge between compartmental models of thyroid hormone kinetics and actual measurement of tissue pools of thyroid hormones in an ectothermic vertebrate. In summary, tissue concentrations of thyroid hormones did not echo plasma concentrations, indicating that thyroidal status cannot be inferred from plasma data alone.

Factors potentially regulating hepatic growth hormone (GH) receptors in coho salmon (Oncorhynchus kisutch) have been investigated. From December to June of the first year, relative changes in hepatic 125I-sGH binding and 35SO4 incorporation by ceratobranchial cartilage were similar. Stunted salmon, which in seawater have elevated plasma GH yet fail to grow, showed lower hepatic 125I-sGH binding than did normally growing seawater salmon. However, MgCl2 treatment of stunts' membranes to reveal total specific binding of 125I-sGH indicated receptor occupation by endogenous sGH. Total specific 125I-sGH binding was low in seawater stunts and remained low if these fish remained unfed after return to fresh water, but increased approximately twofold upon feeding. Total specific binding in fasted salmon in fresh water showed a trend toward decreased levels by 1 week; by 3 weeks, binding was 40% lower than in fed fish. There was a positive correlation (r = 0.600) between condition factor and total specific binding in fed and fasted salmon in fresh water. Two weeks after hypophysectomy total specific binding was 50% lower than in sham-operated control salmon, indicating pituitary regulation of GH receptors. GH treatment reduced both free and total 125I-sGH binding in salmon examined 24 hr after treatment. Treatment with recombinant bovine insulin-like growth factor I, thyroxine, or cortisol did not affect free 125I-sGH binding. Both the pituitary and nutrition appear to be prime regulators of hepatic GH receptors in coho salmon.

Insulin-like growth factor-binding proteins (IGF-BPs) were demonstrated in the circulation of four teleost fish species. In the coho salmon (Oncorhynchus kisutch), serum binding of 125I-labelled human IGF-I (125I-hIGF-I) was competitively inhibited by addition of excess recombinant bovine IGF-I (rbIGF-I) in a manner similar to that when rat serum was used. Western-ligand blot procedure using the same labelled hormone identified at least three major forms of IGF-BPs in the plasma of all four teleost species investigated: coho salmon, striped bass (Morone saxatilis), tilapia (Oreochromis mossambicus), and longjawed mudsucker (Gillichthys mirabilis). The first form is around 40-50 kDa, may be regulated by growth hormone (GH), and seems to be a good candidate for the fish version of mammalian IGF-BP3 (which is in the same size range and is GH-regulated). The second and third forms are 29 kDa and 31 kDa and are good candidates for the fish versions of mammalian IGF-BP1 and IGF-BP2, respectively, as they appear to be regulated by insulin and are in the same size range as their mammalian counterparts. Functionally different classes of circulating IGF-BPs may be conserved between fish and mammal.

The effect of insulin-like growth factor I on growth rate of coho salmon (Oncorhynchus kisutch) was examined. Juvenile coho salmon received implants of osmotic minipumps containing recombinant bovine insulin-like growth factor I (rbIGF-I) or saline for a period of 3 to 4 weeks. High doses of rbIGF-I (greater than 0.13 microgram.g-1.d-1) resulted in hypoglycemia and death. In 2-year-old coho salmon, 0.09 microgram.g-1.d-1 rbIGF-I administered for 25 days doubled linear growth rate and increased growth rate in weight by 40%. In rapidly growing, 1-year-old coho salmon, growth rate was not altered by rbIGF-I at 0.01 or 0.05 micrograms.g-1.d-1 for 31 days. In ration-limited fish exhibiting slow growth in the control group, rbIGF-I (0.02 microgram.g-1.d-1) increased linear growth rate by up to threefold and growth rate in weight by up to fourfold. The results indicate that exogenous treatment with mammalian IGF-I can stimulate coho salmon growth under some conditions, and that endogenous IGF-I may be an important factor in regulating growth of teleosts.

The direct hormonal control of sulfate uptake by cartilage matrix of coho salmon was examined by exposing branchial cartilage to 1 microCi.ml-1 35SO4 for 48 hours at 15 degrees C in a defined medium. Sulfate uptake occurred primarily in cartilage (rather than bone) and the amount of specific uptake was similar in epibranchial and ceratobranchial cartilages. Intact and hypophysectomized coho salmon starved for 22 days had equivalent in vitro sulfate uptake, which in both cases were 30% of the uptake seen in branchial cartilage of fed, intact controls. In branchial cartilage from starved coho salmon, in vitro exposure to recombinant bovine insulin-like growth factor I (rbIGF-I) at 1, 10, 100, and 1,000 ng.ml-1 caused a dose-dependent increase in sulfate uptake, with a maximum 3-fold increase over control at 1,000 ng.ml-1 rbIGF-I. Coho salmon insulin (1, 10, 100, and 1,000 ng.ml-1) resulted in a maximum 30% increase in sulfate uptake at the highest dose. Growth hormone and triiodo-L-thyronine had no direct effect on in vitro sulfate uptake. The results indicate that IGF-I has direct effects on coho salmon cartilage and may be an important regulator of growth in salmon and other teleosts.

Mammary glands from ovariectomised neonatally diethylstilbestrol (DES)-exposed (0.1 microgram daily for the first 5 days of life) mice seem morphologically indistinguishable from those of ovariectomised controls. However, administration of exogenous hormones reveals a differential response. In DES-exposed mice, estrogen implantation resulted in greater incidence of dilated ducts along with greater incidence of dilated ducts along with greater incidence and severity of terminal ductal hyperplasia and greater severity of cystic alveolar adenosis; combined estrogen and progestin treatment resulted in greater severity of terminal duct hyperplasia and less alveolar formation, and progestin treatment resulted in lower incidence and degree of lateral budding. Thus, mammary sensitivity to sex steroids is altered by early exposure of mice to DES.

Newborn female BALB/cCrgl mice receiving 5 micrograms of testosterone or 0.01 micrograms of diethylstilbestrol daily for the first 5 days of life were examined at various times after secondary exposure to testosterone and 17 beta-estradiol, respectively. Neonatal administration of testosterone induced squamous stratification associated with constant cornification of the vaginal epithelium in intact mice. Later exposure to testosterone suppressed cornification, resulting in superficial epithelial mucification in almost all mice by 4 months of age. However, at 6 months of age, the incidence of mucification dropped to 58%. Cervicovaginal lesions developed in the groups of mice given neonatal testosterone in combination with later testosterone and sacrificed at 4 and 6 months of age. Continuous vaginal stratification was found in 14% of ovariectomized, neonatally diethylstilbestrol-treated mice at 13 months of age. The incidence of this ovary-independent change increased to 40% at 24 months of age. Postnatal estrogen replacement significantly increased the incidence of squamous stratification in these mice. Neonatal diethylstilbestrol treatment alone induced cervicovaginal lesions in 4.5% of ovariectomized mice at 13 months of age; secondary 17 beta-estradiol exposure significantly enhanced the development of lesions to 44%. However, at 24 months of age, there was no difference in the incidence of lesions in ovariectomized, neonatally treated mice with or without the secondary 17 beta-estradiol treatment. These results suggest that the effects of neonatal exposure to a relatively low dose of estrogen, androgen, or related substance may become obvious later in life as a result of later exposure to hormones.

Estrogenic potencies of various antiestrogens, including keoxifene (Kx) and trifluoperazine (Tfp), on reproductive tracts of ovariectomized adult mice, and effects of neonatal Kx and Tfp on reproductive organs were studied in C57BL/Tw mice. In adult ovariectomized mice, weight, DNA, and protein of the uterus and vagina were increased by 3 daily injections of 100 micrograms clomiphene, tamoxifen (Tx), and nafoxidine, and of 1 microgram estradiol-17 beta (E), but not by Kx. Antiestrogenic potency of Kx was studied in adult mice given injections of E. Kx significantly suppressed the E-induced increase in weight, DNA, and protein in the uterus and vagina. Tfp (20 micrograms), known as a tranquilizer and an antiestrogen, had no estrogenic effect on either organ. Male and female mice given 5 daily injections of Kx or Tfp from the day of birth were examined at 30, 40, and 60 days of age. Weights of testis, epididymis, and seminal vesicle in neonatally Kx-treated mice were significantly lower than in controls at 30 and 40 days. Spermatozoa were not formed in the seminiferous tubules of Kx-treated mice, although spermatogenesis occurred at 60 days. In neonatally Kx-treated females, weight of the uterus at 60 days and of the vagina at 40 and 60 days was significantly lower than in controls. Corpora lutea were absent from the ovaries of Kx-treated females. In neonatally Tfp-treated mice of both sexes at all ages examined, no differences were found in organ weights or histology, other than lower spermatogenic indices at 40 and 60 days of age.(ABSTRACT TRUNCATED AT 250 WORDS)

In 1987, striped bass (Morone saxatilis) that were nearly dead (moribund) were captured by hand net, and apparently healthy striped bass were caught by hook and line from adjacent waters in the Sacramento-San Joaquin Delta or, alternatively, caught by hook and line from the Pacific Ocean. The livers of these three groups of striped bass were examined for chemical contamination by gas chromatography, by gas chromatography-mass spectrometry, and by immunoassay. Moribund striped bass livers were greatly contaminated by chemicals compared to healthy fish caught in the Delta and the Pacific Ocean. The types of contaminant encountered suggested that industrial, agricultural, and urban pollutants were present in the livers of moribund fish. Although the variability in the amount of hepatic contaminants observed among the groups of fish does not provide direct proof of causation, the large amount of pollutants suggests that chemical contamination (possibly acting as multiple stressors) contributes to the hepatotoxic condition of the moribund striped bass and may lead to an explanation of the die-off in the Sacramento-San Joaquin Delta region.

Neonatal treatment with diethylstilbestrol (DES) induces ovary-independent vaginal epithelial changes in mice. The response of vaginal epithelial cells from intact prepuberal BALB/cCrgl mice treated neonatally with 2 micrograms of DES for 5 days to growth-stimulatory and -inhibitory factors was studied using a serum-free collagen gel culture system that sustains the growth of normal vaginal epithelial cells. Cells from control and DES-exposed mice at 21 days of age showed about a 5-fold increase in number during 10 days in a serum-free medium supplemented with transferrin, bovine serum albumin fraction V, insulin, and epidermal growth factor. Epidermal growth factor and insulin stimulated dose-related proliferation of vaginal epithelial cells from both control and DES-exposed mice; however, cells from DES-exposed mice showed a reduced growth response to epidermal growth factor and an increased growth response to insulin, compared with control cells. Insulin-like growth factor I (1-100 ng/ml) tested in the absence of insulin failed to stimulate cell growth. Transforming growth factor-beta (0.05-5 ng/ml) consistently inhibited cell growth in a dose-dependent manner.

The ability of cortisol to increase gill Na+,K(+)-ATPase activity was examined in several salmonid species during development. Coho salmon (Oncorhynchus kisutch) parr were unresponsive to cortisol in vitro (10 micrograms/ml for 2 days) in November. Responsiveness was significant from January to March, peaking in January just prior to seasonal increases in gill Na+,K(+)-ATPase activity. Gill tissue became unresponsive to in vitro cortisol in April when in vivo gill Na+,K(+)-ATPase activity peaked. The ability of cortisol to stimulate gill, Na+,K(+)-ATPase activity in postemergent fry (2-3 months after hatching) was examined in chum (O. keta), chinook (O. tschawytscha), coho, and Atlantic salmon (Salmo salar). Initial levels of gill Na+,K(+)-ATPase activity were elevated in chum salmon, which normally migrate as fry. Cortisol (10 micrograms/ml for 4 days in vitro) increased gill Na+,K(+)-ATPase activity in chum salmon fry (48% above initial levels), had a limited but significant effect in chinook salmon fry, and had no effect in coho and Atlantic salmon fry. In an in vivo experiment, Atlantic salmon previously exposed to simulated natural photoperiod (SNP) and continuous light (L24) received four cortisol injections of 2 micrograms.g-1 every third day. SNP fish responded with increased gill Na+,K(+)-ATPase activity (+66%), whereas L24 fish were not affected. Atlantic salmon presmolts with initially low levels of gill Na+,K(+)-ATPase activity responded to cortisol in vitro, whereas smolts with initially high levels of gill Na+,K(+)-ATPase activity were unresponsive.(ABSTRACT TRUNCATED AT 250 WORDS)

A serum-free vaginal explant culture system was established to investigate the in vitro effect of estrogen on the growth of mouse vaginal epithelium. Vaginal explants were isolated from 40-day-old, ovariectomized BALB/cCrgl mice and cultured in a basal unsupplemented medium or in basal medium plus various doses of 17 beta-estradiol. Explants were processed for histology at the end of culture periods or were given 4-hour pulses of tritiated thymidine at various times and processed for autoradiography. Vaginal epithelium increased 3- to 5-fold in thickness and 2-fold in the number of epithelial cell layers during 72 hours of culture without estrogen; addition of estrogen did not significantly influence epithelial growth. Keratinization of vaginal epithelium occurred within 48 hours of culture in the absence of estrogen, and again addition of estrogen did not accelerate its appearance. Covering the explants with collagen decreased the estrogen-independent growth of vaginal epithelium. Autoradiography showed that ca. 70-90% of basal epithelial cells entered S phase during the initial 4 hours of culture and that this number declined rapidly after 48 hours to ca. 20%. Addition of 1.8 nM 17 beta-estradiol significantly decreased the labelling index of basal cells at 48 hours, but did not affect the labelling index at 24 and 72 hours. Stromal cells were not labelled at any time. Thus, DNA synthesis, cellular proliferation, and differentiation (keratinization) of vaginal epithelium in organ culture occurred without estrogen and were not stimulated by the addition of estrogen.

Effects of prolactin on morphology and numbers of chloride cells in the opercular membrane of seawater-adapted tilapia (Oreochromis mossambicus) have been examined. Following five daily injections of ovine prolactin at a dose of 10 micrograms.g body wt-1, blood samples were taken and opercular membranes were removed and stained with a fluorescent mitochondrial dye (dimethylaminostyrylethylpyridiniumiodine), a fluorescent derivative of ouabain (anthroylouabain), and a histological stain specific for the extensive tubular system of chloride cells (zinc-osmium-iodine). Mean plasma osmolarity and sodium increased 23-24% following prolactin injection. An increase in the relative frequency of chloride cells between 20 and 180 microns2 in cross-sectional area and a decrease in the relative frequency of chloride cells greater than 180 microns2 were observed following prolactin injections. Average cell size decreased 46-70% and cell height decreased 26-38% following prolactin injections. There was no significant change in cell density. Anthroylouabain staining was observed in both prolactin- and saline-injected fish, and no significant effect on Na+,K(+)-adenosinetriphosphatase activity was seen in either opercular membrane or gill tissue. The results demonstrate an effect of prolactin on chloride cell size and provide a morphological correlate for decreased secretory activity of chloride cells following prolactin injections.

Pregnant ICR/JCL mice were given 4 daily subcutaneous injections of 0.2-2000 micrograms diethylstilbestrol (DES) starting on day 15 of gestation. Offspring of mothers given DES were killed at 1-10 days of age and examined for nodules of enlarged polygonal cells under the epithelium of the Müllerian (upper) vagina. Some offspring were ovariectomised at 30 days and killed at 120 days. The nodules which appeared in the prenatally DES-exposed mice (2-2000 micrograms/day) at 3-7 days were not connected with the epithelium of the sinus vagina and reacted positively to an antibody to epidermal growth factor. Nodule formation may prove to be prodromic of later ovary-independent vaginal changes in the DES-exposed mice. Epithelial stratification (2-2000 micrograms/day) and downgrowths and/or pegs (20-2000 micrograms/day) occurred in vaginae of ovariectomized mice exposed prenatally to DES; however, adenosis-like lesions occurred only in the offspring of mothers given the highest prenatal injections of 2000 micrograms DES. Wolffian remnants and hypospadias (2-2000 micrograms/day) were also encountered in the DES-exposed mice. Ovary-independent stratification of the uterine epithelium (20-2000 micrograms/day) and disorganization of the circular musculature (2-2000 micrograms/day) were also observed in the DES-exposed mice. None of these changes was found in ovariectomised 0.2 micrograms DES-exposed and control mice.

Mouse vaginal epithelial cells were isolated from intact 21-day-old BALB/cCrgl mice and cultured in a serum-free medium (SF20: basal medium supplemented with insulin, epidermal growth factor, transferrin, and bovine serum albumin--fraction V) to examine the proliferation, differentiation, and specificity of estrogen-induced growth retardation in vitro. Histologic and ultrastructural studies showed that vaginal epithelial cells undergo differentiative changes in vitro in the absence of estrogen, and that these changes are similar to those induced in vivo by estrogen. Addition of 17 beta-estradiol inhibited cellular proliferation in a dose-dependent manner. Whereas other estrane derivatives (17 alpha-estradiol and estriol) also significantly retarded cellular proliferation, cholesterol, testosterone, and progesterone had no effect. Keoxifene, an antiestrogen, significantly reversed estrogen-induced growth inhibition, resulting in proliferation of estrogen-treated cells equivalent to that of the untreated control. The results suggest that both proliferation and differentiation of prepubertal mouse vaginal epithelial cells in vitro are estrogen-independent, and that the growth inhibition is a specific estrogen-induced response.

The amino acid sequence of tilapia (Oreochromis mossambicus) growth hormone (GH) was determined directly by Edman degradation of peptide fragments generated by lysyl endopeptidase and trypsin digestion. The N-terminal residue was deduced to be pyroglutamic acid through the use of pyroglutamyl aminopeptidase; its removal allowed amino acid sequence determination of the remainder of the N-terminal trypsin peptide by Edman degradation. Tilapia GH is composed of 187 amino acid residues and shows high similarity to other perciform GHs. Sequence identities are: 89% with tuna GH, 83% with bonito GH, 85% with yellowtail GH, 89% with red sea bream GH, and 34% with bovine GH. The two asparagine residues (Asn-148 and Asn-184) were recovered by Edman degradation, suggesting the absence of N-glycosylation.

Normal endometrial luminal epithelial cells isolated from ovariectomized approximately 40-day-old BALB/cCrgl mice were purified by Percoll density gradient centrifugation and grown as primary cultures in collagen gel matrix and serum-free medium. Cells increased threefold in number during the 9-day culture period. Deletion of insulin, epidermal growth factor or bovine serum albumin resulted in decreased growth. Addition of any single factor to the unsupplemented medium had no effect. Relatively high levels of cytosolic oestrogen receptors and progestin receptors were demonstrable in the cultures. Addition of oestrogen did not enhance epithelial cell proliferation. On the contrary, all doses of oestrogen (180 fmol/l to 218 nmol/l) were inhibitory. Continuous exposure to oestradiol-17 beta (1.8 nmol/l) for 9 days in serum-free medium resulted in a decrease in cytosolic oestrogen receptors with an associated nuclear accumulation of oestrogen receptors. A corresponding increase in cytosolic progestin receptors was also observed, indicating that no qualitative modification of the oestrogen receptor system had occurred. Thus, as previously reported for vaginal epithelial cells, oestrogen, despite its stimulation of specific product synthesis (progestin receptors), did not increase proliferation of endometrial luminal epithelial cells in this culture system.

Binding sites for native chum salmon growth hormone (sGH) in coho salmon (Oncorhynchus kisutch) hepatic membranes were analyzed by radioreceptor assay. Displaceable (specific) binding represented up to 25% of total radiolabeled sGH added. Binding was dependent on buffer pH and membrane protein concentration, and was complete by 24 hours at 15 degrees C. Specific binding was greatest in liver membranes, and was also detected in muscle, ovary, gill, kidney, and brain. Scatchard analyses indicated a single class of hepatic binding sites that were specific for sGH. In stunts, abnormal seawater salmon with elevated plasma GH levels and inhibited growth, specific binding of sGH to liver membranes was three times lower than in normal seawater smolts. The concentration of salmon GH binding sites was decreased in stunt livers by 60%, while their affinity for sGH was unchanged. Down-regulation of hepatic GH receptors by high plasma GH levels may explain in part the low sGH binding in stunts.

Epithelia and fibromuscular walls were dissociated from the vaginae of ovariectomized BALB/cCrgl mice (ca. 41 days old) exposed neonatally to diethylstilbestrol (DES) or sesame oil and purified by centrifugation through Percoll density gradients. Neonatal exposure to DES caused the vaginal epithelium to become permanently proliferated and partially keratinized; the control epithelium was low cuboidal. The major cellular proteins expressed in each tissue compartment were examined by two-dimensional gel electrophoresis of [35S]methionine-labeled tissues. The epithelia and fibromuscular walls displayed distinctive two-dimensional protein patterns. In the DES-exposed vaginal epithelium, the expression of two proteins (one had a molecular size of 65 kDa and a pl of 6.0, and the other had a molecular size of 38 kDa and a pl of 6.3) was increased, while the expression of three proteins with molecular sizes of 25, 30, and 140 kDa and pls of 5.6, 5.6 and 6.7, respectively, was reduced, relative to the control epithelium. In the DES-exposed vaginal fibromuscular walls, the expression of 9 proteins was increased whereas the levels of 21 specific proteins, distinct from those in the epithelium, were decreased. Thus, long-term tissue-specific alterations in the synthesis of a select number of cellular proteins occur in the DES-exposed vagina.

The in vitro S-oxygenation of thiobencarb (Bolero; p-chlorobenzyl N,N-diethylthiocarbamate) in the presence of hepatic microsomes from freshwater- and seawater-adapted striped bass was investigated. Thiobencarb S-oxide was the principal metabolite and accounted for 98% of the total thiobencarb metabolized by striped bass liver microsomes. Studies on the biochemical mechanisms for striped bass hepatic S-oxygenation suggest that this reaction is catalyzed largely by the flavin-containing monooxygenase and to a lesser extent by cytochromes P-450. Following the short incubation period used, no thiobencarb sulfone was detected and no evidence was found for a contribution of cooxidation in the S-oxidation of thiobencarb. This conclusion was supported by studies with microsomes and purified mammalian monooxygenases which also metabolized thiobencarb without cooxidizing factors. Highly purified cytochrome P-450IIB-1 S-oxygenated thiobencarb more efficiently than highly purified hog liver flavin-containing monoxygenase. Thiobencarb S-oxide and thiobencarb sulfone were efficient carbamylating agents and reacted with thiol and amine nucleophiles, whereas thiobencarb itself was relatively stable to transthiocarbamylation. Monooxygenase-catalyzed S-oxygenation of thiobencarb by striped bass liver microsomes may represent a bioactivation process which could explain the known toxicity of thiobencarb in fish.

Neonatal exposure to the synthetic estrogen, diethylstilbestrol, is known to affect the structure of the male reproductive system; thus, changes may also occur in the levels of hormone receptors. Prolactin receptor levels from the reproductive systems of male BALB/c mice exposed neonatally to diethylstilbestrol were analyzed. Neonatal exposure to diethylstilbestrol caused significant decreases (i) in prolactin receptor levels in the seminal vesicle, ductus deferens, and anterior and ventral prostates and (ii) in tissue weight and protein content in reproductive organs other than the ventral prostate.

Growth of anterior and ventral prostatic epithelial cells from mice neonatally exposed to diethylstilbestrol (DES) and from unexposed control mice was compared at different time points in serum-free collagen gel culture. A longer maintenance of the initial plating density (lag in growth) was observed in cultured DES-exposed ventral prostatic cells. Neonatal DES exposure resulted in two colony types: one similar to colonies arising from unexposed cells and one which appears to be non-growing. Keratinization was observed in some DES-exposed anterior prostatic cell colonies. Removal of epidermal growth factor from the serum-free medium significantly decreased growth in 3 of the 4 groups compared with their growth in the complete serum-free medium.

Sustained growth of mouse ventral and anterior prostatic epithelial cells embedded within collagen gel matrix was achieved in a serum-free medium composed of Dulbecco's modified Eagle's medium and Ham's F12 medium, 1:1 (vol/vol), supplemented with bovine serum albumin fraction V, epidermal growth factor, transferrin, cholera toxin, prolactin, 5 alpha-dihydrotestosterone, cortisol, putrescine, fibroblast growth factor, and a trace element mixture. Three-dimensional growth of prostatic epithelial cells occurred inside the collagen gel matrix. This serum-free medium allowed cell growth greater than sevenfold over 10 d in culture. Tissue recombination and cell culture techniques were integrated to demonstrate that cultured cells retained prostatic characteristics. Following 10 d of culture, epithelial colonies from mouse ventral and anterior prostatic epithelial cell cultures were isolated and combined with rat fetal urogenital sinus mesenchyme and grown for 4 wk under the renal capsule of intact athymic male mice. These tissue recombinants showed distinctive prostatic histologic characteristics (alveoli and ducts lined with cuboidal or columnar epithelium surrounded by stroma). When histologic sections of recombinants were stained with the Hoechst 33258, epithelial cells of mouse origin were distinguishable from stromal cells of rat origin.

Female C57BL/Crgl mice were neonatally exposed to various doses of coumestrol to determine the threshold doses required for the occurrence of reproductive tract abnormalities. Newborn mice received daily subcutaneous injections of 10(-3), 10(-2), 8 X 10(-2), 10(-1), 1, 5, 25, 50, and 100 micrograms coumestrol in 0.005 ml dimethyl sulfoxide (DMSO) or DMSO alone, or received no treatment for the first 5 d of life. Some of the animals were ovariectomized at 40 d of age. Mice were killed at 20-22 mo of age. All neonatal doses of coumestrol advanced vaginal opening before that of controls. At 2 and 20-22 mo of age, doses greater than or equal to 25 micrograms consistently resulted in ovary-independent persistent vaginal cornification as judged by vaginal smears. Intact untreated and DMSO-treated control mice exhibited aging changes in the genital tract, some cervical adenosis and early cervicovaginal pegs and downgrowths, uterine cystic glandular hyperplasia, corpora lutea, and scattered areas of ovarian ceroid deposition. Intact mice receiving neonatal coumestrol exhibited cervicovaginal pegs and downgrowths (at all doses with the exception of 25 and 50 micrograms), cervical adenosis (at doses greater than or equal to 8 X 10(-2) micrograms), uterine squamous metaplasia (significant at doses greater than or equal to 50 micrograms), and a decrease in uterine cystic glandular hyperplasia (significant at doses greater than or equal to 25 micrograms). The levels of 10(-1), 5, and 100 micrograms neonatal coumestrol daily resulted in hemorrhagic follicles. An increase in ovarian ceroid deposition (significant at doses greater than or equal to 5 micrograms) was observed. At 40 d and 20-22 mo of age, corpora lutea were consistently absent from the 100-micrograms-treated animals. Most of the ovariectomized untreated and DMSO-treated control animals showed typical castrate-like morphology of the genital tract, with the majority of the control mice exhibiting uterine cystic glandular hyperplasia. Ovariectomized mice receiving coumestrol neonatally exhibited various degrees of cervicovaginal alterations: pegs and downgrowths (significant at all doses with the exception of 10(-1) micrograms), endometrial collagen deposition (significant at greater than or equal to 25 micrograms), and reduced or absent uterine glands (significant at 10(-3), and 10(-11), and at all doses greater than or equal to 5 micrograms).