Occurrence of a new corticotropin-like intermediate lobe peptide in salmon pituitary glands

A new corticotropin-like intermediate lobe peptide (CLIP) has been identified in the pituitary of chum salmon, Oncorhynchus keta. The newly isolated peptide is a tetracosa peptide, which is two residues longer than the predominant form, CLIP I, with the following amino acid sequence, H-ArgProIleLysValTyrAlaSerSerLeuGlu GlyGlyAspSerSerGluGlyThrPheProLeuGlnAlaOH. This peptide, named CLIP II is the fourth line of evidence in the teleost that the pituitary gland secretes two different forms of processed hormones, for which precursor molecules are coded on two separate genes. Together with the structures of α-melanotropin I and II, two putative ACTH molecules are proposed.     

Biosynthesis, processing and release of pro-opiomelanocortin related peptides in the intermediate lobe of the pituitary gland of the frog (Rana ridibunda)

The biosynthesis of pro-opiomelanocortin (POMC) and related peptides by the intermediate lobe of the pituitary gland was studied in the frog Rana ridibunda using the pulse-chase technique. Analysis of radioactive proteins by dodecyl sulfate polyacrylamide gel electrophoresis showed that during pulse incubations a 36,000 dalton (36K) glycosylated prohormone was synthesized. It disappeared slowly during chase incubations, giving rise to another glycosylated protein (Mr 18K), identified as the N-terminal fragment of POMC. This latter protein was secreted to the incubation medium. High performance liquid chromatography analysis of peptides synthesized during chase incubations revealed the biosynthesis of two peptides related to gamma-MSH, three peptides related to alpha-MSH, one endorphin-related and one CLIP-related peptides. These newly synthesized peptides were slowly secreted to the incubation medium. Among the alpha-MSH related peptides, only the des-N alpha-acetyl alpha-MSH form of the peptide was found to be present within the cells, in contrast to the incubation medium where the presence of des-N alpha-acetyl alpha-MSH and a modified alpha-MSH was demonstrated.     

Neuronal intermediate filaments in the developing tongue of the frog Rana esculenta

The expression of several neuronal intermediate filament (NIF) proteins was investigated in the tongue of metamorphosing tadpoles (stage 38-45 of Gosner) and in adult individuals of the frog, Rana esculenta by means of immunohistochemistry. Results showed that nerve fibres at early stages of tongue development expressed peripherin (a NIF protein usually found in differentiating neurones) as well as the light- and medium molecular weight NIF polypeptide subunits (NF-L and NF-M, respectively); in the adult frog, peripherin was still found in nerve fibres reaching the fungiform papilla together with NF-M, but NF-L immunoreactivity was absent therein. Clusters of epithelial cells expressing peripherin were found in the early developing tongue before differentiation of taste organs, and NF-L and NF-H immunoreactivities were present in basal (Merkel) cells of the adult frog taste disc. Results indicate that neurones innervating the adult frog's taste disc maintain a certain plasticity in their cytoskeleton and that neuronal-like cells are present in the undifferentiated and differentiated tongue epithelium possibly playing a role in the developing and mature taste organ.     

Characterization of gonadotropin-releasing hormone (GnRH) binding sites in the pituitary and testis of the frog, Rana esculenta

Frog, Rana esculenta, pituitary and testis gonadotropin-releasing hormone (GnRH) receptors were characterized by using 125I-chicken IIGnRH (cIIGnRH) as radiolabeled ligand. At 4 C equilibrium binding of 125I-cIIGnRH to pituitary homogenates was achieved after 90 min of incubation; binding of 125I-cIIGnRH to testis membrane fractions reached its maximum at 60 min of incubation. Binding of the radioligand was a function of tissue concentration, with a positive correlation over the range 0.5-2 tissue equivalents per tube. One pituitary and one testis per tube were used as standard experimental condition. Incubation of the pituitary homogenate with increasing concentrations of 125I-cIIGnRH indicated saturable binding at radioligand concentrations of 1 nM and above while for the testis membrane preparation saturation was achieved using 5 nM 125I-cIIGnRH. The binding of 125I-cIIGnRH was found to be reversible after addition of the cold analog and the displacement curves could be resolved into one linear component for both tissues. Scatchard analysis suggested the presence of one class of binding sites for both pituitary and testis (Pituitary: Kd = 1.25 +/- 0.14 nM and Bmax = 8.55 +/- 2.72 fmol/mg protein; testis: Kd = 2.23 +/- 0.89 nM and Bmax = 26.48 +/- 7.39 fmol/mg protein). Buserelin displaced the labeled 125I-cIIGnRH with a lower IC50 as compared with cIIGnRH cold standard, while Arg-vasopressin (AVP) was completely ineffective, confirming the specificity of binding.     

Glucocorticoid receptor of frog (Rana esculenta) liver

The presence of a glucocorticoid soluble receptor is demonstrated in frog liver cytosol. The kinetic characterization of frog liver cytosolic receptor for glucocorticoids is reported and its steroid specificity assessed. Results indicate a gross similarity between frog liver and mammalian glucocorticoid receptor, being a major difference the reduced binding capacity.     

Ultrastructure of venom glands in the frog (Rana esculenta)

Electron microscopic study of skin venom glands in the frog, Rana esculenta, revealed the syncytial structure of the inner (secretory) wall which presents two distinct zones: a basal (juxtamuscular) one, which contains nuclei and major cytoplasmic organelles, and an apical one where large electron-dense granules form and accumulate. Granules are seen to arise inside clusters of tightly packed smooth endoplasmic reticulum (SER) elements, which suggests that the SER system is mainly involved in synthesis of this material. A high glutaraldehyde concentration (5%) also reveals a poorly defined material filling the intergranular cytoplasm. No apical limits to the syncytium could be traced, which suggests massive holocrine secretion. Nerves insinuate between the muscle cells and occur all along the internal face of the muscular layer, sometimes in close contact with the syncytium. The gland duct, the wall of which consists of epidermal cells, is blocked, in contact with the gland, by an epidermal bud linked externally to the muscle layer surrounding the gland. Thus, only strong muscle tension such as to expel all or part of the epidermal bud can trigger granule release. This phenomenon can be induced by the subcutaneous injection of epinephrine, but the high and distressing dose needed to provoke appreciable changes in venom glands renders unlikely any natural intense venom release triggered by epinephrine in the frog.     

Nociceptin binding sites in frog (Rana esculenta) brain membranes.

The recently discovered natural heptadecapeptide nociceptin (orphanin FQ) shares some homology with the opioid peptides but it binds to a distinct receptor type, termed nociceptin receptor. This study demonstrates the presence of specific nociceptin recognition sites in brain membrane fractions of an amphibian, Rana esculenta. Para-iodo-Phe(1)-nociceptin-amide was radiolabelled by catalytic dehalotritiation, resulting in p[(3)H]Phe(1)-nociceptin-amide of 25 Ci/mmol specific radioactivity. Specific binding of [(3)H]nociceptin-amide to frog brain membranes was found to be saturable and of high affinity with equilibrium K(d) values in the low nanomolar range. A single set of binding sites with about 180 fmol/mg protein maximal binding capacity was obtained in saturation and competition experiments. [(3)H]Nociceptin-amide binding could easily be inhibited by synthetic nociceptin compounds but not by opioid ligands. Both sodium ions and 5'-guanylylimidodiphosphate decreased the binding of the radioligand by transferring the receptor to a lower affinity state. Nociceptin dose-dependently stimulated the binding of the nonhydrolysable, radiolabeled GTP-analogue guanosine-5'-O-(3-thio)triphosphate ([(35)S]GTPgammaS) to G-proteins in frog brain membranes. Addition of 1 microM naloxone caused no significant change in the curves, indicating that nociceptin-mediated activation of G-proteins occurred through nonopioid mechanism.     

Vitellogenin hormonal control in the green frog, Rana esculenta. Interplay between estradiol and pituitary hormones

1. The effect of estradiol and pituitary hormones on the titre of serum vitellogenin has been studied in Rana esculenta by rocket immunoelectrophoresis. 2. Hepatic synthesis of vitellogenin depends on physiological doses of estradiol. 3. Gonadotrophins enhance the uptake, presumably by acting directly on the oocyte plasma membrane. 4. In addition, our data support direct pituitary intervention on liver synthesis and/or release of vitellogenin. 5. Hormonal response, as evaluated by vitellogenin serum titres, tends to increase from November to July. This could be the expression of a modification, throughout the sexual cycle, of liver sensitivity to the hormones.     

Intratesticular control of spermatogenesis in the frog, Rana esculenta.

Adult intact and hypophysectomized (PDX) frogs, Rana esculenta, were treated with a gonadotropin releasing hormone agonist (GnRHA, HOE 766) and/or cyproterone acetate (CPA), the antiandrogen, in order to investigate the regulation of primary spermatogonial (I SPG) multiplication in vertebrates. Treatment with GnRHA (injections containing 900 ng administered for 12 days on alternate days) caused a significant increase of the mitotic index (MI) of I SPG in PDX animals and a further MI increase of SPG was observed when 0.66 mg CPA was given concomitantly with GnRHA. The treatment with 0.66 mg CPA in combination with GnRHA also increased secondary spermatocyte (II SPC) appearance. Moreover, number of nests containing spermatids (SPT) decreased as CPA, in combination with GnRHA, was administered in increasing doses (0.33 and 0.66 mg/injection). Intact animals treated with CPA (0.66 mg/injection) showed a time-dependent I SPG multiplication increase which reached highest values after 28 days. Secondary SPC also proliferated until day 28; meanwhile the number of nests containing SPT decreased. Neither testosterone nor R5020 (a progestin which is not converted to androgens) modified the basal and GnRHA-induced spermatogonial proliferation. These results confirm that in the frog, Rana esculenta, spermatid formation is impaired by CPA treatment and that I SPG multiplication is enhanced by a direct effect of GnRHA; moreover, we suggest that the absence of spermatids constitutes a signal promoting spermatogonial proliferation.     

Characterization of eight forms of corticotropin-like intermediary lobe peptide from the rat intermediary pituitary

The isolation and characterization of eight forms of corticotropin-like intermediary lobe peptide (CLIP, adrenocorticotropin18-39) from the intermediary lobe of the rat pituitary has been accomplished by using reversed phase high performance liquid chromatography. The eight forms are the result of all combinations of the presence or absence of three post-translational modifications. These are glycosylation, phosphorylation, and removal of the carboxyl-terminal amino acid. The sites of phosphorylation and glycosylation are at serine 31 and asparagine 29, respectively. The eight forms (in order of elution from the reversed high performance liquid chromatography column) are glycosylated, phosphorylated CLIP18-38; glycosylated, nonphosphorylated CLIP18-38; nonglycosylated, phosphorylated CLIP18-38; nonglycosylated, nonphosphorylated CLIP18-38; glycosylated, phosphorylated CLIP18-39; glycosylated, nonphosphorylated CLIP18-39; nonglycosylated, phosphorylated CLIP18-39; and nonglycosylated, nonphosphorylated CLIP18-39.     

Tyrosine aminotransferase activity of frog (Rana esculenta) liver

1. The presence of tyrosine aminotransferase is reported both in particulate and soluble fractions of frog liver. 2. The activity of the soluble enzyme of frog liver was investigated with regard to its dose and time dependence, its substrate specificity and concentration dependence, its thermal sensitivity as well as pH and temperature dependence. 3. It appears that the properties of the soluble tyrosine aminotransferase of frog liver are in close agreement with those reported for the mammalian liver enzyme.     

Effect of tunicamycin on biosynthesis, processing and release of proopiomelanocortin-derived peptides in the intermediate lobe of the frog Rana ridibunda

The intermediate lobe of the pituitary gland synthesizes a glycoprotein, proopiomelanocortin (POMC), which is cleaved by specific proteolytic enzymes to generate several hormonal peptides. The purpose of the present study was to examine the possible role of the carbohydrate moiety in the synthesis, intracellular processing and release of POMC-derived peptides in frog (Rana ridibunda) intermediate lobe cells. In vitro incorporation of [3H]-labelled glucosamine gave rise to three major radioactive products. Trypsin digestion of each of these glycopeptides gave a single glucosamine-labelled tryptic fragment with identical chromatographic characteristics. We conclude that Rana POMC is glycosylated in only one site (its gamma-MSH region) and that intracellular processing of this prohormone gives rise to smaller glycopeptides including glycosylated gamma-MSH. Treatment with the antibiotic tunicamycin (10 micrograms/ml, 6 hr) inhibited the glycosylation of POMC but did not significantly alter the neosynthesis of the peptide moiety of the precursor. Pulse-chase experiments combined with high-performance liquid chromatography analysis of the peptides derived from POMC revealed that inhibition of glycosylation by tunicamycin had no effect on the enzymatic cleavage of the precursor nor on the release of mature peptides. Thus, it is concluded that, in the frog, glycosylation of POMC has no influence on the biosynthesis, processing and release of intermediate lobe hormones.     

Circannual variations in mevalonate utilization in frog (Rana esculenta)

1. The fate of mevalonate, the product of HMGCoA reductase, was studied in male and female frogs (Rana esculenta) in order to explain the circannual variations of enzyme activity. 2. The incorporation of 2-14C MVA into unsaponifiable lipids, cholesterol and dolichol in liver, plasma and eggs was followed. 3. Labeled MVA shows a different utilization depending on season and sex. In spring and summer cholesterol synthesis is related to hepatic reserve storage in both sexes, while the peak of enzyme activity, present only in females in fall, seems committed to cholesterol export into the blood and uptake by the oocytes. 4. The presence of a MVA-derived protein identifiable with vitellogenin and labeled on the lipid moiety, suggests that HMGCoA reductase activity in fall is committed to the lipidation of this protein essential for oocyte maturation.     

Ovarian activity and reproduction in the frog, Rana esculenta

Rana esculenta specimens were collected, during the last 13 years, in well-defined areas around Naples. The annual ovarian cycle shows distinct phases of recrudescence (starting September; vitellogenesis), breeding (late March-early July; egg deposition and active oogenesis) and quiescence (July-August; no follicular growth). Previtellogenic follicles are recruited for vitellogenesis in early September and in between two successive ovulatory waves. Breeding congregations are generally formed after a heavy rain fall and eggs are laid in standing waters, temporary or permanent. A maximum of three clutches of eggs is produced during the breeding season, at roughly monthly intervals. All mature females reproduce to some extent. Ovarian weight and clutch size are positively correlated to body weight. Depending upon the body size, the potential clutch size ranges from 1000 to 3500 eggs during the first wave of ovulation and it is notably smaller in the successive wave(s) of ovulation. Egg masses and tadpoles are left unprotected and mortality is high. The life cycle from the fertilized egg to completion of metamorphosis is 2 months and oogenesis in the ovary starts in the larva before the onset of metamorphic climax. Young females hatching from the first clutch of eggs may reach sexual maturity and breed in May the following year; those hatching from the last clutch require nearly 20 months to reach sexual maturity. The importance of some endocrine and exocrine factors for the regulation of ovarian activity and reproduction is discussed.     

Hepatic respiratory compensation and blood volume in the frog (Rana esculenta)

In the frog, Rana esculenta (L.), the liver can change in volume by over 25%, depending on the respiratory conditions of the animal: in well-oxygenated specimens the organ can hoard about half of the total amount of erythrocytes in its sinusoids, and release them into the bloodstream under conditions of hypoxia. This phenomenon can be observed at a temperature of 6°C by comparing the liver volumes and haematic values of chlorobutanol-anaesthetized animals exposed to the air or submerged in still water (a condition which induces hypoxia): the blood volume remains constant, at about 5 ml per 100g of body weight, but red blood cell count and haematocrit value differ by as much as 50%. At 18°C there is an increase in oxygen demand and in anaesthetized animals, which rely totally on cutaneous respiration, the compensatory liver mechanism can no longer be observed, since all the available erythrocytes are already circulating in a blood volume which, depending on respiratory conditions, can vary between about 7 and 8ml/100g. At 30°C, cutaneous respiration alone does not allow the anaesthetized animals to survive long enough to stabilize their haematic parameters.