Effects of postnatal treatment with naloxone on plasma gonadotropin,prolactin, testosterone and testicular functions in male rats

Opioid peptides are implicated in the control of gonadotropin and prolactin secretion. The role of opioid antagonist naloxone and its effects on plasma gonadotropin, prolactin, testosterone levels and testicular hyaluronidase, acid phosphatase, [³H]uridine and thymidine incorporation, RNA, DNA and protein concentrations were evaluated in rats after administration of naloxone beginning day 1 through 21 and autopsied on 45, 60 and 90 days of age. Plasma gonadotropin and testosterone levels were significantly elevated after naloxone treatment. Testicular hyaluronidase and acid phosphatase activity increased till 60 days post treatment and declined thereafter. Concentrations of RNA and protein did not change significantly but the concentration of DNA declined at 45 and 60 days of age. These results suggest that endogenous opioid peptides exert regulatory influence on gonadotropin secretion which in turn control the testicular function in the male rat.     

Relative ability of ovine follicle stimulating hormone and itsβ-subunit to generate antibodies having bioneutralization potential in nonhuman primates

The relative ability of ovine follicle stimulating hormone and itsβ-subunit, two potential candidates for male contraceptive vaccine, to generate antibodies in monkeys capable of bioneutralizing follicle stimulating hormone was assessed usingin vitro model systems. Antiserum against native ovine follicle stimulating hormone was found to be highly specific to the intact form with no cross-reactivity with either of the two subunits while the antiserum againstβ-subunit of follicle stimulating hormone could bind to theβ-subunit in its free form as well as when it is combined withα-subunit to form the intact hormone. Both antisera could block the binding of the hormone to the receptor if the hormone was preincubated with the antibody. However, the follicle stimulating hormoneβ-antisera could only inhibit the binding of the hormone partially (33% inhibition) if the antibody and receptor were mixed prior to the addition of the hormone, while antisera to the native follicle stimulating hormone could block the binding completely (100% inhibition) in the same experiment. Similarly antisera to the native follicle stimulating hormone was significantly effective in blocking (100%) response to follicle stimulating hormone but not theβ-subunit antisera (0%) as checked using anin vitro granulosa cell system. Thus the probability of obtaining antibodies of greater bioneutralization potential is much higher if intact hormone is used as an antigen rather than itsβ-subunit as a vaccine. Majority of the work reported here was carried out during the tenure of Visiting Scientist fellowship awarded by the MRC Canada to the first author.     

Localization of corticotropin-releasing hormone (CRF)-like immunoreactivity in the central nervous system of the elasmobranch fish,Scyliorhinus canicula

Summary The occurrence and localization of immunoreactive corticotropin-releasing factor (CRF) in the brain and pituitary of the elasmobranch fish Scyliorhinus canicula, were studied by means of specific radioimmunoassay and immunohistochemistry using the indirect immunofluorescence method. Brain and pituitary extracts showed a good cross-reactivity with the ovine CRF antiserum, but serial dilutions of tissue samples did not completely parallel the standard curve. Relatively high concentrations of CRF-like material were found within the pituitary, diencephalon, and telencephalon. CRF-like immunoreactive perikarya were observed in the preoptic nucleus and in the nucleus lateralis tuberis. Numerous immunoreactive cells appeared to be of the CSF-contacting type. CRF-like immunopositive fibers were seen to run through the hypothalamus within the ventro-medial floor of the infundibular region. A dense plexus of immunoreactive nerve endings terminated in the median eminence and the neurointermediate lobe of the pituitary. These results indicate that a neurosecretory system containing CRF-like immunoreactivity exists in the brain of elasmobranchs, a group of vertebrates which has diverged early from the evolutionary line leading to mammals. In addition, our data support the notion that a CRF-like molecule is involved in the regulation of corticotropic and melanotropic cell activity in this primitive species of fish.     

Effects of a juvenile hormone analogue on the eggs, post-embryonic development, metamorphosis and diapause induction of the Colorado potato beetle, Leptinotarsa decemlineata

The effect of a juvenile hormone analogue, S-71639, was tested on the eggs, four larval instars and adults of the Colorado potato beetle, Leptinotarsa decemlineata Say, by topical application or after treatment of the foodplant. The last larval instar is very sensitive to S-71639. Treatment of this instar delayed the onset of pupation and prevented adult emergence. Treated animals showed severe abnormalities, but they were not immediately killed at the doses used in this study. Treatment of larvae also interfered with the photoperiodic induction of diapause. Adults, kept under diapausing conditions, started to lay eggs after treatment with S-71639. The ovicidal effect of the compound was rather weak. The implications for practical use of S-71639 in control of the Colorado potato beetle are being discussed.

---

Résumé L'effet d'un analogue de l'hormone juvénile, S-71639, a été testé sur les oeufs, les quatre stades larvaires et les adultes du doryphore, Leptinotarsa decemlineata Say, par application topique ou après traitement de la plante-hôte. Le dernier stade larvaire est très sensible au S-71639. Le traitement de ce stade retarde le début de la métamorphose et empêche l'émergence adulte. Les animaux traités montrent de graves anomalies, mais ne sont pas immédiatement tués par les doses utilisées dans cette étude. Le traitement des larves perturbe aussi l'induction photopériodique de la diapause. Les adultes placés dans des conditions de diapause, commencent à pondre après traitement au S-71639. L'effet ovicide de la substance est plutôt faible. Les implications pour l'utilisation pratique du S-71639 dans la lutte contre le doryphore sont discutées.

     

Solubilization and characterization of a lactogenic receptor from human placental chorion membranes

Prolactin has a wide range of actions, including osmoregulation and the control of mammary gland development and lactation. These effects are mediated through a high-affinity cell surface receptor, which has been well characterized in a number of animal tissues. The molecular characteristics of the human receptor are unknown, however. The present studies were initiated, therefore, to determine the binding and molecular characteristics of the lactogenic receptor of human placental chorion membranes. Subcellular fractionation studies showed that the bulk of the receptor sedimented in the microsomal fraction at 45,000gav. Endogenous ligand was dissociated from the receptor with 3.5 M MgCl2 or 0.05 M acetate buffer (pH 4.8) with preservation of binding activity. The microsomal receptor bound human growth hormone (hGH), human prolactin (hPRL), ovine prolactin (oPRL), and human placental lactogen (hPL) but not non-primate growth hormones, indicating a narrow specificity for lactogenic hormones. The binding was only partially reversible in agreement with the known binding kinetics of animal lactogenic receptors. The receptor was solubilized with 45% yield from the microsomes using 16 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) detergent-250 mM NaCl, and the binding activity was fully restored by a two-fold dilution in the binding reaction to reveal a KD of 0.8 nM for hGH and a binding capacity of 200 fmol of specifically bound hGH per mg of microsomal protein. Gel filtration chromatography indicated the minimum molecular weight of the ligand-receptor complex was approximately 60,000 daltons, and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of covalently cross-linked 125I-hGH-receptor complexes revealed a molecular size of 58,000 daltons. When account was taken of the contribution of the ligand, a molecular weight of 36,000 for the receptor's binding domain was obtained. These data indicate that the chorion lactogenic receptor has very similar binding and molecular characteristics to the lactogenic receptors from other mammalian species. Chorion membranes are thus a convenient source of material for the further purification and characterization of the human lactogenic receptor.     

Multiple episodes of induced secretion of human growth hormone from recombinant AtT-20 cells

Recombinant AtT-20 cells expressing human growth hormone (hGH) secreted the hormone at a constant, basal rate of 0.3–0.5 ng/10⁵ cells-hour when exposed to medium without secretagogues. When triggered with 8 bromo-cyclic AMP, cells secreted hGH at an initial rate of 1.7 ng/10⁵ cells-hour while intracellular hGH declined sharply. Upon extended exposure to secretagogue, secretion decreased gradually to the basal rate and intracellular hGH stabilized at a value 40% the initial. In cells switched from secretion to growth medium, the total rate of hGH accumulation intracellularly and in medium was 2.2 times that observed with cells never exposed to secretagogue; however, only a fraction of the hormone was stored intracellularly and the rest was secreted. When cells were exposed alternately to growth and secretion medium, induced cells secreted at rates at least two times higher than uninduced controls during the first five cycles. The induced response deteriorated with time, however, in parallel with outgrowth of attached cells by foci of round cells, and by the eighth cycle induced secretion did not occur. Operational modifications that may improve the performance of cycling schemes are discussed.     

Structural studies of nerve terminals containing melanin-concentrating hormone in the eel,Anguilla anguilla

Summary Eels were adapted to black- or white-coloured backgrounds and the pituitary glands were prepared for light and electron microscopy. Immunocytochemical staining was used to study the distribution of the neurohypophysial melanin-concentrating hormone in the neurointermediate lobe. The hormone was located in small, elliptical, electron-opaque neurosecretory granules, measuring approximately 120×90 nm. The neurones terminated on blood vessels in the centre of the neurohypophysis and on the basement membrane separating neural and intermediate lobe tissues. The results of both light and electron immunocytochemistry and of radioimmunoassay are consistent with a higher rate of hormone release from eels adapted to white backgrounds than from those adapted to black backgrounds. In addition to this, when fish that had been adapted to white tanks were transferred to black tanks, there was an accumulation of irMCH in the gland and an increased numerical density of secretory granules at nerve terminals. These results reinforce the proposal that MCH is released during adaptation to a white background, to cause melanin concentration and to inhibit MSH release, and that its release is halted in black-adapted fish.     

Immunocytochemical demonstration of the neurosecretory systems containing putative moult-inhibiting hormone and hyperglycemic hormone in the eyestalk of brachyuran crustaceans

Summary By use of antisera raised against purified moultinhibiting (MIH) and crustacean hyperglycemic hormone (CHH) from Carcinus maenas, complete and distinct neurosecretory pathways for both hormones were demonstrated with the PAP and immunofluorescence technique. By double staining, employing a combination of silver-enhanced immunogold labelling and PAP, both antigens could be visualized in the same section. Immunoreactive structures were studied in Carcinus maenas, Liocarcinus puber, Cancer pagurus, Uca pugilator and Maja squinado. They were only observed in the X-organ sinus gland (SG) system of the eyestalks and consisted of MIH-positive perikarya, which were dispersed among the more numerous CHH-positive perikarya of the medulla terminalis X-organ (XO). The MIH-positive neurons form branching collateral plexuses adjacent to the XO and axons that are arranged around the CHH-positive central axon bundle of the principal XO-SG tract. In the SG, MIH-positive axon profiles and terminals, clustered around hemolymph lacunae, are distributed between the more abundant CHH-positive axon profiles and terminals. Colocalisation of MIH and CHH was never observed. The gross morphology of both neurosecretory systems was similar in all species examined, however, in U. pugilator and M. squinado immunostaining for MIH was relatively faint unless higher concentrations of antiserum were used. Possible reasons for this phenomenon as well as observed moult cycle-related differences in immunostaining are discussed.     

The synthetic precursor specific region of pre-pro-parathyroid hormone forms ion channels in lipid bilayers

We have used the chemically synthesized sequence of pre-pro-parathyroid hormone and several of its analogues to test the notion that the capacity of amphipathic peptides to aggregate in membranes and form ion-permeable channels correlates with their ability to function as signal sequences for secreted proteins. We found that pre-pro-parathyroid hormone (the signal sequence and pro-region of parathyroid hormone (M)), as well as some of its analogues, forms aggregates of monomers which are ion-permeable. The ion-permeable aggregates (2–3 monomers) formed by (M) are voltage-dependent and are more permeable for cations than for anions. The compounds which formed ion channels in bilayers also acted as potential signal sequences. We conclude that the ability of peptides to form ion-permeable pathways in bilayers may be correlated to their ability to function as signal peptides.     

The central nervous system of Bulla gouldiana: peptide localization

In the present study, we describe the structure of the central nervous system (CNS) of the marine gastropod Bulla gouldiana, and compare it with the structure of the CNS of the related mollusc, Aplysia californica. In addition, we performed an immunohistochemical analysis of a series of peptides, and the synaptic vesicle protein, synapsin I, in the central nervous system of B. gouldiana. The most common peptide in the B. gouldiana nervous system is the molluscan cardioexcitatory peptide (FMRFamide), which is present in a significant proportion of B. gouldiana neurons. A smaller number of neurons exhibit immunoreactivity to antisera raised against the calcitonin gene related peptide, vasopressin, vasoactive intestinal peptide, cholecystokinin, galanin and enkephalin. In some instances there is colocalization of two or more peptides. Very few neurons or axons exhibit synapsin I-like immunoreactivity. The patterns of immunoreactivity to these antisera is quite similar to the patterns that have been described in other gastropods, including Lymnaea stagnalis and Aplysia californica. These observations emphasize the importance of FMRFamide-like compounds in phylogenetically old nervous systems and indicate that compounds similar to mammalian peptides are present in the gastropod. Thus, the production of a wide variety of peptide molecules and their use in neuronal function appears to be a highly conserved phylogenetic process.