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Urotensin II, a peptide hormone from the caudal neurosecretory system of the teleost, Gillichthys mirabilis, was isolated by using classical chromatographic techniques and high-performance liquid chromatography (HPLC). Direct microtechniques for sequence determination were used to establish its structure. Urotensin II from Gillichthys is a 1363-dalton dodecapeptide with the amino acid sequence Ala-Gly-Thr-Ala-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val. This sequence is homologous with somatostatin in positions 1 and 2 and 7-9. The sequence has been verified by the production of a bioactive synthetic urotensin II. The possible chemical and physiological significance of its homology to somatostatin is discussed.

We have investigated the capacity of vaginal and uterine epithelia (VE and UE) to reexpress normal morphology and hormone responsiveness following cell culture. VE and UE from adult ovariectomized mice were grown in a collagen gel matrix with serum-free medium for 7-10 days. Proliferation of these cells occurs in the absence of 17 beta-estradiol and is not stimulated by 17 beta-estradiol; the VE usually does not keratinize or stratify in vitro. Cultured VE and UE were recombined with homologous vaginal or uterine stroma (VS and US, respectively) and these recombinants were grown under the renal capsule of female hosts for 4 weeks. The epithelium of the VS + VE recombinants cycled, proliferated and stratified in response to estrogen and mucified normally in response to progesterone. The UE that was grown in vivo with cultured US also showed normal morphology and estrogen responsiveness. These changes in the UE and VE were not simply a result of return to the in vivo environment, as epithelia alone in collagen gels transplanted under the renal capsule did not survive. These results indicate that both VE and UE, which are not estrogen-dependent in vitro, reexpress their estrogen dependency and normal morphology when recombined with homologous stroma and grown in vivo. Thus, the changes these cells show when grown in culture are the result of altered conditions in vitro rather than an irreversible alteration in the cells themselves or the selection of specific subpopulations that are not mitogenically or morphologically responsive to estrogen under any condition.

Neonatal treatment of female mice with oestrogen or androgen induces persistent vaginal cornification, which may be dependent or independent of ovarian hormones according to the amount administered. The concentration of oestrogen-binding protein in the cytoplasm and in the nuclear fraction was measured in the uterus and vagina of mice treated neonatally with either steroid alone or in combination with prolactin. Vaginal oestrogen responsiveness (dependence) of mice treated with low and high doses of steroids was correlated with oestrogen receptor content. Neonatal treatment with prolactin in combination with the low dosage steroid treatments favoured development of ovary-independent cornification and a concomitant decrease in oestrogen receptors in about 50% of the mice studied.

Sodium-potassium-activated adenosine triphosphatase (Na-K-ATPase) activity increased in the urinary bladder of the euryhaline teleost Platichthys stellatusafter transfer from sea-water to fresh water. This increase also occurred after injection of prolactin into seawater Platichthys, simulating the results of freshwater transfer. In Kareius bicoloratus, which does not survive transfer to fresh water, prolactin does not increase bladder Na-K-ATPase activity. The differences in response of these two species to prolactin may be related to the degree of their euryhalinity. There may be a relationship between adaptability to fresh water and responsiveness of bladder Na-K-ATPase to prolactin.

The histopathogenesis and growth behavior of mammary tumors and dysplasias induced by a single intragastric dose of 7,12-dimethylbenz[a]anthracene in 50-day-old virgin female Lewis rats were examined both in situ and after transplantation into gland-free mammary fat pads of syngeneic hosts. Terminal mammary ductules are indicated as a site of origin of both ovarian hormone-dependent mammary tumors and spontaneously gegressing mammary tumors, and terminal ductule hyperplasia appears to be an early stage in mammary tumor formation. The precancerous nature of hyperplastic alveolar nodules induced by dimethylbenzanthracene in rats has been further examined, and our studies indicate that these nodules are not significantly preneoplastic.

The complete amino acid sequences of a pair of tilapia (Oreochromis mossambicus) prolactins (PRLs) were determined. The larger PRL of molecular mass 20,836 Da consists of 188 amino acid residues. The smaller PRL of molecular mass 19,584 Da is 11 residues shorter. On alignment of the two sequences, the 19.6-kDa PRL (tPRL177) has two conspicuous deletions on the NH2-terminal side of the disulfide bond which connects the first and second cysteine residues. The degree of similarity between the two PRL sequences is unexpectedly low (130 identical residues, 69%) compared with that between the variants of other teleostean PRLs. Circular dichroism spectra and hydropathy profiles suggest structural similarity of the two PRLs. The sequence of the 20.8-kDa PRL (tPRL188) has 69% identity with that of salmon PRL. The sequence of tPRL177 is 56% identical with that of salmon PRL. Each tilapia PRL is equally similar to mammalian PRLs (about 30% identical residues). Regions highly conserved among teleostean and mammalian PRLs were identified on the COOH-terminal side of the disulfide bond connecting the first and second cysteine residues.

These studies are concerned with the structural and functional evolution of the ancestrally related pituitary prolactins and somatotrophins. Prolactin-like biological activities of human somatotrophin (hGH) and its peptide fragments were bioassayed in vitroon the mouse mammary gland and the teleost urinary bladder. Plasmin modified-hGH was as active as hGH in both bioassays. The NH2-terminal 134-residue fragment possessed about 10% of the lactogenic and urinary bladder potency of hGH, whereas the CO2H-terminal 51-residue fragment was inactive at the concentrations observed. These results suggest that the same regions of primary structure are responsible for the prolactin-like actions of hGH on the target organs of lower and higher vertebrates. Alteration of the tertiary structure of hGH, human chorionic somatomammotrophin, and ovine prolactin by performic acid oxidation destroys the mammary gland activities of these hormones.

Sustained growth in primary culture of vaginal epithelial cells from ovariectomized adult BALB/cCrgl mice embedded within or seeded on collagen gel matrix was achieved in a serum-free medium composed of Ham's F-12 medium/Dulbecco's modified Eagle's medium, 1:1 (vol/vol), supplemented with insulin, bovine serum albumin fraction V, epidermal growth factor, cholera toxin, and transferrin. Three-dimensional growth of vaginal epithelial cells occurred inside the collagen gel matrix. Cell numbers increased 4- to 8-fold in collagen gel and about 4-fold on collagen gel after 9-10 days in culture. The effect of 17 beta-estradiol (0.00018-180 nM in gel or 0.018-180 nM on gel) and diethylstilbestrol (DES; 0.0186-186 nM in gel) on the growth of vaginal epithelial cells was examined. The addition of estrogen did not enhance the growth of vaginal epithelial cells during this time period either in the complete medium or in a suboptimal medium. Cultures on floating collagen gels in the serum-free medium are composed of 1-3 cell layers with superficial cornification. Estrogen does not appear to be a direct mitogen for vaginal epithelial cells, at least in this system.

The pituitary of the cichlid fish tilapia secretes two prolactins (PRLs) of molecular masses 20 kDa and 24 kDa. The 20-kDa PRL has an isoelectric point in the range of those of mammalian PRLs (pI 6.7), but the 24-kDa PRL is unusually basic (pI 8.7). Partial sequence information indicates that the PRLs are homologous but distinct proteins, differing by five amino acids within the first 29 NH2-terminal residues. Homology in the known region is higher with chum salmon PRL than with known mammalian PRLs. Reversed-phase HPLC permits isolation of these two PRLs and a single tilapia growth hormone from culture medium or from the pituitary in a single step. HPLC and radio-HPLC analysis of [3H]leucine pulse-chase experiments reveal that each PRL is secreted in vitro at remarkably high rates (21 pmol per gland per hr) and that the two PRLs are released in approximately equimolar amounts, suggesting the coordinate regulation of the secretion. Both PRLs exert characteristic PRL activity in that they prevent the loss of Na+ from the plasma of hypophysectomized tilapia in fresh water.

We have evaluated the effect of neonatal administration of mouse prolactin (PRL) antiserum on the developmental expression of T- and B-lymphocytes in the thymus and spleen of female BALB/c mice. Newborn female mice were injected subcutaneously with a 50-microliters aliquot of PRL antiserum or normal rabbit serum on days 1, 2, and 3. On neonatal day 5, the PRL antiserum-treated group had a significantly (P less than 0.05) increased population of cells in the thymus and the spleen that were positive for Thy-1.2 and for L3T4. Increases in Thy-1.2- and L3T4-positive cells in the thymus were detectable also on days 8 and 14 in mice that received the PRL antiserum and in mice injected with bromocriptine, a dopamine agonist that inhibits PRL release from the anterior pituitary. On neonatal days 21, 28, and 32, there were no significant differences in the percentage of cells positive for Thy-1.2, Ly-2 (formerly Lyt-2), or L3T4 antigens in the thymus. However, there were significant increases in the percentage of Thy-1.2- and L3T4-positive spleen cells in the bromocriptine-treated group at all times monitored and in the PRL antiserum-treated group except on day 14. In addition, the percentage of splenocytes that were positive for IgG was significantly increased in the PRL antiserum-treatment group on days 8-28, although not on neonatal day 32. Of tissues known to contain PRL receptors, neonatal administration of PRL antiserum or bromocriptine resulted in a significant alteration in the wet weight of spleen and liver, with no significant effect in thymus, heart, and kidney. Pituitary implants also resulted in a significant increase in both concanavalin A- and lipopolysaccharide-stimulated thymidine incorporation into murine splenic lymphocytes prepared from 45-day-old female mice. These data extend the role of PRL as an immunomodulator of adult lymphocyte function to a role in the developmental expression of T- and B-lymphocyte populations in the thymus and spleen of mice.

Mucosal fluid, sodium and chloride transfer were measured in everted sacs of rat, guinea-pig and hamster jejunum, and in rat ileum and colon, and in guinea-pig gall bladder. After treatment of the animal with ovine prolactin, a highly significant enhancement of fluid and NaCl absorption was observed in rat, hamster and guinea-pig jejunum. Prolactin treatment caused a significant increase in fluid and NaCl transfer in rat ileum, but not in guinea-pig ileum or rat colon. Prolactin administration had no consistent effect on fluid and NaCl absorption by the guinea-pig gall bladder. The several regions of the mammalian gut appear to differ in their responsiveness to prolactin.

Isometric tension responses to neuropeptides were recorded from anococcygeus muscles isolated from male mice. This smooth muscle tissue is innervated by inhibitory nonadrenergic, noncholinergic nerves that resemble, ultrastructurally, the peptidergic neurons of the gastrointestinal tract; the physiological function of the anococcygeus is not known. Slow sustained contractions were produced by oxytocin (0.2-20 nM), [Arg8]vasopressin (0.4-200 nM), and [Arg]-vasotocin (0.4-100 nM); the mouse anococcygeus is, therefore, one of the few examples of nonvascular smooth muscle from male mammals to respond to low concentrations of oxytocin and related peptides. Substance P (0.5-8 microM) caused distinctive, biphasic increases in muscle tone of some, but not all, preparations. Other neuropeptides producing contractions were neurotensin (2-100 microM) and thyrotropin-releasing hormone (2-100 microM); the responses were of similar time course and displayed selective cross-desensitization, suggesting that these two peptides act through a common distinct mechanism. Tetradecapeptide somatostatin (10-80 microM) and its analog urotensin II (0.1-5 microM), a dodecapeptide from the urophysis of the teleost fish Gillichthys mirabilis, produced similar slowly developing relaxations of carbachol-induced tone. Piscine urotensin II, of which there are no reported effects on nonvascular mammalian systems, was 20-50 times more potent than somatostatin, a well-established mammalian hormone. Of the peptides studied, only vasoactive intestinal polypeptide (0.05-1 microM) caused rapid powerful relaxations in low concentrations; this is consistent with its proposed involvement in nonadrenergic, noncholinergic neurotransmission in the mouse anococcygeus.

Ovine prolactin injected subcutaneously for 2 days stimulated an increase in fluid uptake in vitroby everted gut sacs from male Sprague—Dawley rats. Four intestinal segments from each rat were incubated, but only two of the four segments tested showed a prolactin-stimulated increase in fluid transfer in intact rats. In adrenalectomized—nephrectomized rats all four sacs showed an increased fluid transfer after prolactin treatment. Sacs incubated for 1 h in glucose—Ringer solution containing 0·1 mg ovine prolactin/ml did not show an increased water transfer as compared with controls incubated in medium containing albumin. Dietary changes seemed to alter the sensitivity of the gut to prolactin.

Intact female BALB/cCrgl mice, 3–4 weeks old, were pretreated with oestrogen and progesterone for 9 days. Whole mammary glands from these mice were cultivated for 5 days in a synthetic medium supplemented with aldosterone (A), prolactin (MH) and insulin (I), with and without thyroxine (T4) at concentrations ranging from 0·01 to 5 μg./ml.A medium containing 1 μg. A +5 μg.MH +5 μg.I/ml. was generally optimal for lobulo-alveolar development. Addition of thyroxine to this combination resulted in a decrease in development which was highly significant at higher concentrations. However, when cultures were maintained in media containing suboptimal or low amounts of prolactin (1 μg. A + 3 μg. MH +5 μg. I/ml. and 1 μg. A + 1 μg. MH +5 μg. I/ml., respectively), the results indicate two possible effects of thyroxine: lower amounts of thyroxine had synergistic effects, whereas greater amounts had antagonistic effects on lobulo-alveolar development.