Lipovitellin constitutes the protein backbone of glycoproteins involved in sperm-egg interaction in the amphibian Discoglossus pictus.

Our knowledge of the molecules that interact with sperm at the egg membrane is restricted to a short list. In the eggs of Discoglossus pictus, fusion with sperm is limited to a differentiated structure, the dimple, offering several advantages for detecting molecules involved in fertilization. Previous studies have identified fucosylated glycoproteins of 200, 260, and 270 kDa located at the surface of the dimple that are able to bind sperm in vitro. Here, we show that dimple glycoproteins and a protein represented by a 120-kDa band released following gel-into-gel SDS-PAGE of both glycoproteins share the same N-terminal amino acid sequence, which itself is similar to the N-termini of Xenopus liver-synthesized vitellogenin (VTG) and the lipovitellin 1. MALDI/MS mass spectrometry indicated that the 120-kDa band is part of both gps 200 and 270/260. A 117-kDa major protein of the egg lysate exhibits the same MALDI/MS spectrum, and LC-MSMS indicates that this is a lipovitellin 1 (DpLIV) that coincides with the 120-kDa band and is responsible for the formation of the 200-270-kDa dimers. Therefore, lipovitellin 1 constitutes the protein backbone of the dimple glycoconjugates. In vitro assays using polystyrene beads coated with DpLIV or with its dimers indicate that significant sperm binding occurs only with DpLIV dimers. In amphibians, VTG is taken up by the oocyte, where it releases lipovitellins destined to form yolk. In Discoglossus, our data suggest that yolk proteins are also synthesized by the oocyte. The dimple forms in the ovulated oocyte following the exocytosis of vesicles that likely expose DpLIVs at their membrane. Indeed, in whole mounts of immunostained eggs, anti-vitellogenin antibodies label only the surface of the dimple.

Partial cloning of CB1 cDNA and CB1 mRNA changes in stress responses in the Solea solea.

Endogenous cannabinoids, through the CB1 receptor, are involved in the control of several functions including stress responses. The aim of this study was to investigate the presence of cannabinoid receptor CB1 in the sole ovary by partial cloning of brain CB1 cDNA; in a stress paradigm of disturbance by handling, which consisted in catching, netting and hand-sorting, changes of CB1 mRNA were related with those of proopiomelanocortin (POMC) mRNA; the trend and timing of stress responses and adaptation were monitored by measuring plasma cortisol levels. We characterized two forms of CB1-like receptor, termed CB1A and CB1B. The two sole CB1 (both 799bp) share 76% identity in their cDNAs, and the deduced amino acid sequences are 80% identical. The handling stress induced a sustained increase in plasma cortisol levels 1h after the handling began and decreased to low levels 12h after initiation of handling, showing the same trend of ovarian POMC mRNA expression. In addition, while CB1A mRNA did not show any significant changes during handling stress, significantly lower levels of CB1B mRNA were found in stressed fish 1h after the beginning of handling, with CB1 expression increased 24h after stress induction, both in the ovary and brain. It can be concluded that endocannabinoid system is involved in the modulation of adaptive responses to environmental conditions.

Variation of the genetic expression pattern after exposure to estradiol-17beta and 4-nonylphenol in male zebrafish (Danio rerio).

There is much concern about the increasing presence in the environment of synthetic chemicals that are able to disrupt the endocrine system. Among these compounds, 4-nonylphenol (4-NP) is one of the most studied xenoestrogens, due to its widespread accumulation in water sediment and consequent presence in fatty acid of aquatic organisms. Here, we have used a zebrafish microarray representing 16,399 genes to study the effects of 4-NP and estradiol-17beta (E2) in adult male zebrafish in order to elucidate the mechanism of action of 4-NP compared with that of E2. The microarray results showed that both 4-NP and E2 induced a strong expression of vitellogenin (VTG), the sex related precursor of the yolk proteins in oviparous vertebrates. Both treatments induced elevated protein turnover upregulating genes involved in proteolysis and those that are constituents of the ribosome. Many genes regulated by 4-NP and E2 are involved in energy metabolism, oxidative stress defense mechanisms, xenobiotic metabolism, and lipid metabolism. A different pattern of expression in the two treatments was found for genes involved in oxidative stress, since E2 seems to induce the mechanism of detoxification, while 4-NP seems to inhibit this protective mechanism of the cell. Overall, these findings demonstrate that the microarray approach can contribute significantly to the understanding of expression patterns induced by E2 and 4-NP in male zebrafish. The results also demonstrate that 4-NP is able to act through an alternative pattern to that of estradiol-17beta, modulating the expression of the same genes in a different manner.

Assessment of water pollution in the Tronto River (Italy) by applying useful biomarkers in the fish model Carassius auratus.

The Tronto River (southern Marche region of central Italy) is located in an area with neighboring industrial activities and is contaminated with domestic and industrial wastewater. Water quality data analyses revealed the presence of a mixture of low levels of heavy metals and organic compounds. The effects of long-term exposure to Tronto River water on juvenile Carassius auratus were evaluated with an integrated approach using xenoestrogens biomarkers, such as vitellogenin (VTG) and ER beta-1 mRNA expression, and stress parameters (i.e., cortisol and glucose in the blood and glycogen in the liver). Treatment with Tronto River water did not induce VTG synthesis in fish and did not affect ER beta-1 mRNA expression. Moreover, cortisol titers found in the plasma of fish exposed to Tronto River water were lower than those found in the control group. Regarding energy parameters, treatment with Tronto River water induced an increase in plasma glucose and a depletion of liver glycogen reserves.The effects of Tronto River water were studied in parallel with those of 4-NP and CdCl(2). The 4-NP at the dose of 22 microg/L induced the synthesis of peripheral vitellogenin and increase of ER beta-1 titers; on the contrary, CdCl2 exposure at the concentration of 22 microg/L did not induce significant changes on plasma VTG and/or hepatic ER beta-1 levels. In addition, no significant changes in plasma cortisol levels in fish exposed to 4-NP or CdCl(2) were found. Fish exposed to CdCl(2) displayed liver glycogen depletion, but no significant increase in plasma glucose was observed. On the contrary, a 30-day exposure to 4-NP induced only a slight decrease of glycogen reserves without any changes in plasma glucose levels. In conclusion, our study demonstrated that long-term exposure of juvenile goldfish to the water of the Tronto River significantly affects both stress and energy parameters. There is evidence that pollutants, present in Tronto River water, were not able to induce xenoestrogenic effects but caused a functional impairment of the hypothalamum-pituitary-interrenal axis.

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

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.

In-vitro effects of mammalian and amphibian prolactins on hepatic vitellogenin synthesis in Rana esculenta.

Male and female Rana esculenta liver was induced in an in-vitro system by homologous and Rana catesbeiana pituitary to synthesize and release vitellogenin, a lipoglycophosphoprotein precursor of yolk proteins, lipovitellins and phosvitins, in oviparous vertebrates. In the present experiments, the action of prolactin on hepatic vitellogenin synthesis and release was investigated, using ovine prolactin and Rana catesbeiana prolactin. The effects of prolactin on hepatic vitellogenin synthesis displayed different trends related to sex; male liver was found to be more responsive than female liver to both ovine and frog prolactin; more-over, the response to prolactin was dose-related (r = 0.998; P < 0.05) in male but not in female liver. In both sexes, a high degree of seasonality in the responsiveness of the liver was found, since the vitellogenin levels induced by prolactin during the winter phase were significantly (P < 0.001) higher than those produced during the summer phase. Thus, there was no significant difference between the action of ovine and frog prolactin on vitellogenin synthesis; in fact, mammalian prolactins are structurally similar with regard to nucleotide and amino acid sequences. The direct action of prolactin on hepatic vitellogenin synthesis in the frog Rana esculenta is discussed, on the basis of the role played by prolactin as an important growth modulatory hormone in fetal and adult tissues.

Opioids and testicular activity in the frog, Rana esculenta.

The presence and activity of brain, pituitary and testicular beta-endorphin (beta-EP)-like material have been studied in the frog, Rana esculenta, using reverse-phase high-pressure liquid chromatography, coupled with radioimmunoassay and immunocytochemistry. In-vivo and in-vitro treatments with naltrexone were carried out to assess the putative physiological activity of opioid peptides. beta-EP(1-31) and (1-27), together with their acetylated forms, have been identified in brain, pituitary and testis. In particular, beta-EP(1-31) concentrations peaked during July in the brain and pituitary, whilst in testes maximum concentrations were found in April and November. beta-EP immunoreactivity was present in the brain within the nucleus preopticus and nucleus infundibularis ventralis while positive fibres in the retrochiasmatic regions projected to the median eminence. In the testis, interstitial cells, canaliculi of the efferent system, spermatogonia and spermatocytes showed positive immunostaining for beta-EP. In intact animals, naltrexone treatment increased plasma and testicular androgen levels and this effect was confirmed in in-vitro incubations of minced testes. Naltrexone also induced a significant increase in germ cell degeneration. Our results indicated that an opioid system modulates the hypothalamus-pituitary-gonadal axis in the frog, Rana esculenta and, for the first time, we have shown that the testicular activity of a non-mammalian species may be regulated by opiates locally.

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.

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.

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.

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.

Involvement of tyrosine kinase and cAMP in growth hormone-induced vitellogenin synthesis in the anuran, Rana esculenta.

In the frog Rana esculenta, a multihormonal control of vitellogenin synthesis was previously demonstrated. Now in this study, the identity of intracellular second messengers that mediate the GH effects on hepatic VTG synthesis are described. The results clearly indicate that the effect of GH on frog hepatocytes, in vitro, works through a local production of IGF I; in fact, IGF I affects VTG synthesis and its action occurs faster with respect to that of GH. The effect of IGF I was abolished by the anti-estrogen tamoxifen, indicating the involvement of estrogen receptor in VTG induction by IGF I. Furthermore, in vitro treatment of frog hepatocytes with GH resulted in an increase of cAMP with maximum levels after 20 min of treatment. Besides the increase of cAMP, GH induced the appearance of a new phosphotyrosine protein at 20 min, suggesting the occurrence of tyrosine kinase activation. Addition of adenylate cyclase or protein tyrosine kinase inhibitors completely abolished the induction of VTG synthesis, indicating the involvement of cAMP and of a phosphotyrosine protein in VTG synthesis stimulated by both GH and IGF I.

Ovarian opioids and the reproductive cycle of the frog Rana esculenta.

In mammals, proopiomelanocortin-related peptides are involved in reproductive processes both at the hypothalamo-pituitary and ovarian levels. Using immunocytochemical, biochemical and physiological "in vitro" studies, we provide here evidence for a diffuse POMC-related opioid system in the frog Rana esculenta. Ovarian beta-endorphin (beta-EP) is expressed in thecal cells and changes during the reproductive cycle in an inverse relationship with follicular development. Seasonal changes in the ovary are different to those in the brain or in the pituitary. The ratio of acetylated vs native beta-EP in the ovary also changes over the reproductive period, affecting the biological activity of the peptide. During both the reproductive spring period and the summer post-reproductive phase pMol amounts of beta-EP stimulate follicular androgen secretion in vitro, in a naloxone-reversible way. In either period, an inhibition of estradiol, possibly mediated via other factors, is the result of opioid action. In conclusion, these data demonstrate for the first time the widespread presence of beta-EP-related peptides in the frog Rana esculenta. Both immunocytochemical and biochemical evidence, as well as in vitro responses, support a physiological role for beta-EP in ovarian seasonality during the reproductive cycle of this amphibian.