Alberta Maria Polzonetti-Magni

Changes of lysosomal enzyme activities in sea bass (Dicentrarchus labrax) eggs and developing embryos

The sea bass Dicentrarchus labrax is a pelagic egg spawner; sinking eggs are unable to develop into embryos, and this is a limitation in the controlled reproduction of this species. The eggs were divided into good and poor quality, by virtue of their ability to float or sink in seawater. High levels of cathepsins B, D, and L were detected in the eggs, whereas no cathepsin A, C, and E activity was detected. Cathepsin D was found at significantly higher levels in sinking eggs, whereas cathepsin L was more abundant in floating eggs. Since degradation of yolk proteins is essential for the early development of the embryo, the levels of cathepsins A, B, C, D, E, and L were tested in different stages of embryo development. Cathepsin A activity was detectable from the morula stage at which time cathepsin B activity reached its maximal level. Cathepsins A and L reached maximal activity during segmentation, and this corresponded with major changes in the electrophoretic pattern of yolk proteins during embryogenesis suggesting their involvement in yolk protein mobilization at this time. Cathepsin D reached its maximal activity during hatching.

Multihormonal control of vitellogenesis in lower vertebrates.

The comparative approach on how and when vitellogenesis occurs in the diverse reproductive strategies displayed by aquatic and terrestrial lower vertebrates is presented in this chapter; moreover, attention has been paid to the multihormonal control of hepatic vitellogenin synthesis as it is related to seasonal changes and to vitellogenin use by growing oocytes. The hormonal mechanisms regulating vitellogenin synthesis are also considered, and the effects of environmental estrogens on the feminization process in wildlife and humans have been reported. It is then considered how fundamental nonmammalian models appear to be, for vitellogenesis research, addressed to clarifying the yolkless egg and the evolution of eutherian viviparity.

Analysis of Endocrine Disruption in Southern California Coastal Fish Using an Aquatic Multispecies Microarray

Background Endocrine disruptors include plasticizers, pesticides, detergents, and pharmaceuticals. Turbot and other flatfish are used to characterize the presence of chemicals in the marine environment. Unfortunately, there are relatively few genes of turbot and other flatfish in GenBank, which limits the use of molecular tools such as microarrays and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to study disruption of endocrine responses in sentinel fish captured by regulatory agencies. Objectives We fabricated a multigene cross-species microarray as a diagnostic tool to screen the effects of environmental chemicals in fish, for which there is minimal genomic information. The array included genes that are involved in the actions of adrenal and sex steroids, thyroid hormone, and xenobiotic responses. This microarray will provide a sensitive tool for screening for the presence of chemicals with adverse effects on endocrine responses in coastal fish species. Methods We used a custom multispecies microarray to study gene expression in wild hornyhead turbot (Pleuronichthys verticalis) collected from polluted and clean coastal waters and in laboratory male zebrafish (Danio rerio) after exposure to estradiol and 4-nonylphenol. We measured gene-specific expression in turbot liver by qRT-PCR and correlated it to microarray data. Results Microarray and qRT-PCR analyses of livers from turbot collected from polluted areas revealed altered gene expression profiles compared with those from nonaffected areas. Conclusions The agreement between the array data and qRT-PCR analyses validates this multispecies microarray. The microarray measurement of gene expression in zebrafish, which are phylogenetically distant from turbot, indicates that this multispecies microarray will be useful for measuring endocrine responses in other fish.

Variation of the genetic expression pattern after exposure to estradiol-17β 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.

Plasma vitellogenin and 17β-estradiol levels during the annual reproductive cycle of Podarcis s. sicula Raf

Plasma vitellogenin and 17 beta-estradiol concentration were determined during the annual reproductive cycle of the female lizard Podarcis s. sicula Raf. living around Naples. Plasma vitellogenin was purified from estrogenized males for characterization and to raise specific immune serum. Using ELISA, plasma vitellogenin titers were determined in relation to ovary weight; plasma 17 beta-estradiol was measured by RIA method. Native vitellogenin was present as two polypeptide bands: alpha and beta. The electrophoretic patterns, studied in normal male and estrogenized male and female, showed vitellogenin to be a protein present in female and in estrogenized male plasma but not in normal males. Lizard monomeric VTG, determined by SDS-PAGE, was about 200 kDa. Correlations between seasonal ovarian weight variations and plasma vitellogenin and 17 beta-estradiol suggest that ovarian development in Podarcis depends on plasma vitellogenin synthesis, which in turn relies on plasma estradiol levels. The two ovulatory waves observed in this study coincided with the two peak values of plasma vitellogenin and 17 beta-estradiol.

Differential Splicing of Three Gonadotropin-Releasing Hormone Transcripts in the Ovary of Seabream (Sparus aurata)

Abstract Previous studies demonstrated the presence of high-affinity GnRH binding sites and compounds with GnRH-like activity in the ovary of seabream, Sparus aurata, providing evidence for the role of GnRH as a paracrine/autocrine regulator of ovarian function in this species. In the present study, the expression of three forms of GnRH (salmon, chicken-II, and seabream) genes in this marine teleost species was demonstrated for the first time. Moreover, there is evidence for differential splicing and intronic expression of cGnRH-II and sbGnRH. Treatment of seabream follicle-enclosed oocytes with salmon GnRH stimulated reinitiation of oocyte meiosis, whereas chicken GnRH-II treatment was without effect. Novel information was also provided about organization of cGnRH-II and seabream GnRH transcripts, confirming that GnRH gene organization is maintained through evolution, despite changes in the size and sequence of exons and introns.

Molecular components related to egg viability in the gilthead sea bream, Sparus aurata.

In pelagic egg spawners, the production of large numbers of sinking eggs, unable to develop into embryos, represents one of the major limiting factors in controlled reproduction. The aim of this study is to elucidate the molecular differences between floating and nonfloating eggs at cytoplasmic and nuclear level. Comparison of analyses between floating and nonfloating sea bream Sparus aurata eggs evidenced differences in vitelline envelope protein components, such differences being probably related with the hydration process but not with fertilization as supported by the assessment of DNA that doubled after in vitro insemination. These data clearly indicated that the absence of embryo development in nonfloating eggs is not due to lack of fertilization. The cytoplasmic composition was also different, the number of protein components being higher in floating eggs, and these extra components may generate the appropriate osmotic pressure at the base of the hydration process. Some lysosomal enzymes, such as cathepsin D and L both involved in yolk proteolysis, in virgin nonfloating eggs were significantly higher with respect to floating ones; the levels of these two enzymes significantly increased in the latter after fertilization. On the contrary, in nonfloating eggs cathepsin L significantly decreased after fertilization. These changes may be related with a series of metabolic processes vital for the production of viable offspring. The capacity of egg transcription and the protein synthesis in these two types of eggs, indicated by the RNA/DNA and RNA/protein ratios, evidenced that the status of cell transcription rate and protein synthesis capacity is significantly higher in floating eggs. This, in turn, suggested that the lack of embryo development may be due to low levels of proteins involved in cell cycle regulation. Mol. Reprod. Dev. 58:330–335, 2001.

Peptide pheromones in newts

This article reviews the current state of understanding of reproductive pheromones in amphibians, focusing mainly on the purification and characterization of peptide pheromones in newts of the genus Cynops, molecular cloning of cDNAs encoding the pheromone molecules, and hormonal control of secretion of these pheromones. Pheromones that attract sexually developed female Cynops pyrrhogaster and C. ensicauda newts were isolated from the male abdominal glands. The C. pyrrhogaster and C. ensicauda pheromones are peptides, designated sodefrin and silefrin, with the amino acid sequences SIPSKDALLK and SILSKDAQLK, respectively. Each pheromone attracts only conspecific females. Molecular cloning of cDNAs encoding sodefrin and silefrin revealed the presence of precursor proteins that are considered to generate these pheromone peptides. Pheromone precursor mRNA levels and radioimmunoassayable pheromone concentrations in the abdominal glands were elevated by prolactin and androgen. Sexual dimorphism and hormone dependency of the responsiveness of vomeronasal epithelium to sodefrin were noted. Significance of pheromones in the form of peptide for those performing reproductive behavior in an aquatic environment was also discussed.

Acetyl Salmon Endorphin-Like and Interrenal Stress Response in Male Gilthead Sea Bream, Sparus aurata

The present study investigates the role of melanotrope proopiomelanocortin-derived peptide in the interrenal stress response to different stressors in male gilthead sea bream, Sparus aurata. Plasma cortisol and acetyl salmon endorphin (acetyl s-EP), as well as pituitary acetyl s-EP contents, were measured during two stress paradigms: (a) long-term (1-month) confinement and crowding, and (b) short-term (60-min) confinement, crowding, and manipulation. In addition, naltrexone, a highly specific opioid receptor antagonist, was employed in some experimental groups to evaluate the adaptability of the opioid response to interrenal stress. In the long-term (1-month) confinement and crowding, higher plasma cortisol levels and acetyl s-EP concentrations than in the control group were found. However, although plasma cortisol levels significantly increased in both types of stress paradigm, a significant rise in plasma acetyl s-EP was observed only in the case of confinement plus crowding. These data seem to suggest a direct correlation of acetyl s-EP plasma levels exclusively in cases of specific stress, and support previous observations about the different nature of the pituitary-interrenal stress response in salmonids and in mammals. The results obtained in the short-term (60-min) experiments demonstrate the double activation of both the opioid and corticotrope systems when manipulation plus crowding was applied.

Sex steroid profile and plasma vitellogenin during the annual reproductive cycle of the crested newt (Triturus carnifex laur.)

The annual reproductive cycle of the crested newt, Triturus carnifex, has been studied in the field. Temperatures, rainfall, humidity, and photoperiod were recorded throughout the year. Adult male and female newts were sampled monthly; snout vent lengths, crest heights of males, and body ovarian, oviducal, testicular, and abdominal gland weights were recorded. Plasma samples were assayed for androgen, estradiol-17 beta, and progesterone by radioimmunoassay and for vitellogenin by enzyme-linked immunosorbent assay. Air, deep water, water surface, and soil temperatures were low from October to March, but increased in April and May without consistent summer variations. Ovarian and oviducal weights increased in October to reach maximum values between January and March (reproductive period). Crest height and abdominal gland weight in males mirrored the ovarian and oviducal pattern, while testicular weights were maximal in October and November. In females, plasma androgens were high during the reproductive period, and plasma estradiol peaked sharply in March, while plasma progesterone changed little. In the males plasma androgen and estradiol concentrations were similar to those of females, while plasma progesterone was significantly correlated with the cycle in testicular weight. In both sexes androgens showed a significantly negative correlation with air and water surface temperature. Plasma vitellogenin peaked in March but it did not correlate with either ovarian weight or plasma estradiol concentrations. These data support and confirm those previously reported for newts under laboratory conditions. The negative correlation between androgens and temperature suggests that this hormone may trigger the reproductive process. Moreover the correlations between plasma progesterone and testicular weight may indicate that this hormone is involved in male newts reproduction.(ABSTRACT TRUNCATED AT 250 WORDS)