A. Polzonetti-Magni

Multihormonal control of vitellogenin mRNA expression in the liver of frog, Rana esculenta

In Rana esculenta in an in vitro system, hepatic vitellogenin synthesis can be induced by growth hormone in both sexes. In this study: (1) the ability of this hormone to induce transcription of the VTG gene was determined, and (2) this ability was compared with that of estradiol-17 beta. The results indicate that growth hormone stimulates VTG mRNA transcription both in vivo and in vitro, in both sexes. The levels of mRNA are related to protein levels in the medium. In addition, seasonal variation occurs in the VTG gene transcription under growth hormone and estradiol-17 beta; indeed the more active inducer was growth hormone during the reproductive period and estradiol-17 beta during the preproductive phase.

DETECTION OF GNRH MOLECULAR FORMS IN BRAINS AND GONADS OF THE CRESTED NEWT, TRITURUS CARNIFEX

Gonadotrophin-releasing hormone (GnRH) immunoreactivity is detectable in the brain, ovary, and testis of the newt, Triturus carnifex, collected during February (reproductive phase), May, and July (nonreproductive phase). In the brain of May animals, chicken GnRH-II positive cell bodies are located within the terminal nerve, the anterior preoptic area, and the preoptic nucleus, which appears to be devoid of immunoreactive mammalian GnRH cell bodies. During February and July, both chicken GnRH-II and mammalian GnRH are detected only within the terminal nerve and anterior preoptic area. Generally, in the reproductive as well as the nonreproductive periods, chicken GnRH-II fibers are widely distributed in the brain; however, the distribution of fibers of both molecular forms suggests that they exert hypophysiotropic activity. High-pressure liquid chromatography (HPLC) coupled with radioimmunoassay indicates the presence of an early-eluting GnRH peak in brains and gonads but not in plasma. Using chicken GnRH-II antiserum, immunoreactivity is observed in spermatocytes, spermatozoa, and the external theca layer. Seasonal changes of the GnRH-like material are observed in both sexes, and its high concentration detectable during February is in good correlation with the timing of reproduction.

Ovarian melanotropic peptides and adaptation in two teleostean species: Sparus aurata L. and Dicentrarchus labrax L.

The ovarian tissue of Dicentrarchus labrax and Sparus aurata displays two immunoreactive peaks that correspond to the elution time of human des-acetyl alpha-MSH [ACTH(1-13)-amide] and human alpha-MSH. In view of the close identity between the primary structure of fish and human alpha-MSH, these data demonstrate that two MSH-related peptides are present both in sea bream and sea bass ovary. alpha-MSH-like immunoreactivity was found within both granulosa and thecal layers of mature follicles, as well as in the cytoplasm of oogonia of sea bream and sea bass ovary. Gonadal content of ACTH(1-13)-amide and alpha-MSH display differences with regard to season, showing the highest peptide levels in reproductive animals. Moreover, the alpha-MSH content is significantly higher in the ovary of fish farm animals, whereas that of ACTH(1-13)-amide prevails in wild fish ovary.

Detection and localization of gonadotrophin-releasing hormone (GnRH)-like material in the frog, Rana esculenta, ovary.

GnRH-like material has been identified using HPLC followed by RIA in the ovary of Rana esculenta. During the reproductive cycle three immunoreactive GnRH peaks were eluted. One of them coeluted with s-GnRH, the other two forms between GnRH and cII-GnRH. During the recovery phase s-GnRH immunoreactivity disappears. By immunocytochemistry, cII-GnRH immunostaining was localized to granulosa cells while s-GnRH was present in the perinuclear zone of the oocytes.

Occurrence of β-endorphin binding sites in the pituitary of the frog Rana esculenta: effect of β-endorphin on luteinizing hormone secretion

Abstract The possible effect of proopiomelanocortin-derived peptide, β-endorphin on frog gonadotrope cells was investigated. Binding and internalization of β-endorphin to pituitary pars distalis cultured cells were visualized by immunofluorescence and analyzed by means of confocal laser scanning microscopy. Using biotinylated endorphin, the time-course of β-binding showed that this opioid was internalized through receptor-mediated endocytosis, the mechanism in which actin and clathrin were involved; then, the lysosomal degradation program occurred at later stages. The β-endorphin binding was well antagonized by Naloxone, the opiate receptor antagonist, and up-regulated since more rapid response was obtained in the previously primed cells. The double immunostaining reaction for β-endorphin and LH β-subunit revealed that half the β-endorphin labeled cell population was positively immunostained for LH β-subunit, and β-endorphin was able to induce an increasing trend of LH secretion in cultured pars distalis cells. Therefore, it seems that β-endorphin acts directly on pituitary pars distalis and influences gonadotropin secretion through the interaction with its own receptor.

Occurrence of immunoreactive Met- and Leu-enkephalin-like peptides in the ovary of the green frog, Rana esculenta

In the present study, we have localized for the first time Met- and Leu-enkephalin-like material in the ovary of the anuran, Rana esculenta, using the indirect immunofluorescence method. The ovaries were sampled during the main representative phases of the annual reproductive cycle of the frog, living in a mountain pond (Colfiorito, Umbria at 820 m a.s.l.). Strong immunoreactivity to Met- and Leu-enkephalin antisera was observed in the follicle cells of the granulosa layer of vitellogenic oocytes; moreover, during this phase, immunofluorescent materials were also radially localized in the cytoplasm and in the perinuclear zone. The mature oocytes showed Met- and Leu-enkephalin-like immunostaining in the thecal layer and in several granules scattered in the peripheral zone of the yolk. The different pattern of Leu- and Met-enkephalin-like immunoreactivity in the frog ovary parallels and complements the changes occurring in the reproductive (May) and in the vitellogenetic (September) phases during the ovarian cycle. Consequently, these findings strongly support the hypothesis for a local synthesis of these peptides in the ovary and suggest their possible involvement in the control of ovarian function.

Acetyl salmon endorphin-like immunoreactivity in the ovary of two teleostean species: Changes with environmental conditions

The presence of salmon acetylated endorphin (acetyl sEP) in the ovary of seabream and sea bass was investigated through immunocytochemical and biochemical techniques in order to compare aquatic species with terrestrial ones. Endorphin-like immunoreactivity was found in the cytoplasm of oogonia and similar immunostaining was present in the granulosa layer of mature follicles. In both pituitary and ovarian extracts of the two teleostean species, acetyl sEP-like immunoreactivity was distributed over three main peaks, the second one corresponding to the elution time of the reference synthetic peptide. Serial dilutions of HPLC fraction II of the ovaries of both fishes ran parallel with the standard curve obtained with reference peptide. The ovarian content of acetyl sEP, obtained by calculating the integrated area of the fraction II peak, indicates large and highly significant (p < 0.01) differences in the amount of peptide found in ovarian tissues of wild seabream in comparison with that of farmed fish. Increased peptide values in wild animals with respect to farmed fish were also found in the sea bass. These data indicate that not only the pituitary, but also the ovary is sensitive to environmental cues, and strongly suggest the role of opioid peptides in adaptation.

Expression of proopiomelanocortin and its cleavage enzyme genes in Rana esculenta and Xenopus laevis gonads

Abstract: Proopiomelanocortin (POMC) is the precursor protein of different hormones and neuropeptides, and the POMC‐derived peptides are produced through proteolytic cleavage. Prohormone convertase PC1 and PC2 are enzymes responsible for the cleavage of the POMC prohormone. The coexpression of POMC, PC1, and PC2 genes was previously described in the brain and the pituitary gland of Rana esculenta and Xenopus laevis, but no data are available for the gonad. The present work demonstrates a gonadal POMC convertase gene expression in Rana esculenta and Xenopus laevis.