Gilberto Mosconi

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.

EFFECTS OF CAPTIVITY STRESS ON PLASMA STEROID LEVELS IN THE GREEN FROG, RANA ESCULENTA, DURING THE ANNUAL REPRODUCTIVE CYCLE

Abstract 1. 1. Androgen, 17β-estradiol and corticosterone levels were measured by RIA in plasma of Rana esculenta frogs sampled in the field and in the laboratory. 2. 2. Androgen and 17β-estradiol annual cycles of frogs sampled in the field mirrored those of animals sampled in the laboratory 24 hr after capture; on the contrary the absolute values were not similar. 3. 3. 17β-estradiol levels showed a peak 6 hr after capture in the pre-reproduction and reproduction periods. 4. 4. Corticosterone levels increased 72 hr after capture.

Identification and spatial distribution of the mRNA encoding the gp49 component of the gilthead sea bream, Sparus aurata, egg envelope

A cDNA encoding the precursor of one of the major components of gilthead sea bream, Sparus aurata, egg envelope has been cloned by reverse transcriptase polymerase chain reaction (RT‐PCR) techniques. The clone was isolated starting from total RNA extracted from the liver of spawning female fish and estradiol‐17β–treated male fish. Sequence analysis revealed that the cDNA encoded a protein of 405 aa corresponding to 49‐kDa component (termed gp49), a glycoprotein belonging to the N‐linked type. The gp49 protein is homologous to the ZI‐3 of medaka Oryzias latipes, the mammalian ZPC and ZPC homologues of Xenopus laevis (xlZPC) and carp Cyprinus carpio (ccZPC). In addition, the open reading frame also encodes an additional aa sequence, the signal peptide, located in the N‐terminal region of the protein.

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.

Changes in the electrophoretic pattern of yolk proteins during vitellogenesis in the gilthead sea bream Sparus aurata L.

Abstract 1. 1. To some extent, oocyte growth within a follicle is due, as well as to the accumulation of normal cytoplasmic components, to that of the cortical alveoli, and of hepatic-derived protein (vitellogenins). 2. 2. Yolk proteins of pre-maturational oocytes at different stages and ovulated eggs were compared by SDS-gel electrophoresis. The largest components stained by Coomassie Blue and those stained by Stains-all, which had formed during vitellogenesis, either disappeared or diminished, whilst smaller components appeared. 3. 3. The distinct changes in yolk-protein banding patterns during oocyte maturation are suggestive of extensive secondary proteolysis of yolk proteins.

Hormonal control of the IGF system in the sea bream ovary

Abstract: Numerous studies have described the presence of an intragonadal IGF system involved in regulation of gametogenesis in teleost fish. In the present study, the in vivo effects of estradiol‐17β (E2) and growth hormone (GH) exposure on IGF‐I, IGF‐II, IGF1R, and IGFBP2 gene expression in sea bream ovary were monitored by RT‐PCR during prereproductive and reproductive periods. The evidence demonstrates that both hormones investigated here affect the ovarian IGF system, showing that it is not only under GH control, but also can be regulated by sexual hormones; this hormonal modulation is related to reproductive phase.

Diplodus sargus interrenal–pituitary response: chemical communication in stressed fish

In the present study the response of white sea-bream (Diplodus sargus), used as experimental model, to different stocking densities was tested to assess whether the induced stress conditions were able to activate a chemical communication. Once a good recovery was evident, six days after capture and transportation, experiments started to evaluate eventual changes in cortisol plasma levels as well as in both plasma and pituitary alpha-melanocyte stimulating hormone (alpha-MSH) levels. These studies demonstrated that a low stocking density (2.5kg/1000L) is not deleterious for this species while a higher one (10kg/1000L) induces the activation of the hypothalamus-pituitary-interrenal axis (HPI). Thus, in these fish were evident both a significant increase of cortisol plasma levels and a significant decrease of alpha-MSH pituitary levels, while no significant changes were detected in alpha-MSH plasma concentrations. The most relevant result reported in this study, for the first time in marine species, is the presence of a chemical communication among stressed fish. It also should be noted that the data obtained from cortisol and alpha-MSH detections strongly indicate a gender specificity of this chemical signal.