Carlos Frederico Ceccon Lanes

Evaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus

Sperm mediated gene transfer (SMGT) has been successfully used in mammals, amphibians, birds, and some invertebrates. In fish, this methodology has failed or had poor efficiency for the production of transgenic specimens, presumably because the processes regulating the interaction between spermatozoa and exogenous DNA are not well understood. Therefore, the objective was to develop a SMGT protocol for the Brazilian flounder Paralichthys orbignyanus, with an emphasis on the role of seminal plasma DNase on exogenous DNA uptake by fish spermatozoa. In this study, there was strong DNase activity in the seminal plasma of P. orbignyanus; however, this DNase activity was decreased or eliminated by washing the spermatozoa with solutions containing EDTA (DNase activity was completely inhibited by 40 mM EDTA). Three washing solutions were tested, all of which maintained sperm quality. Moreover, it was determined that the no more than 50 ng of exogenous DNA/10(6) cells should be used for SMGT in fish. Finally, it was demonstrated that fish spermatozoa were capable of spontaneous uptake of exogenous DNA after elimination of DNase activity; this was confirmed by exogenous DNA amplification (PCR using sperm genomic DNA as a template) after DNase I treatment. We concluded that whereas DNase activity was an important obstacle for exogenous DNA uptake by fish spermatozoa; controlling this activity improved the efficiency of SMGT in fish.

Identification, tissue distribution and evaluation of brain neuropeptide Y gene expression in the Brazilian flounder Paralichthys orbignyanus.

Neuropeptide Y (NPY) is one of the most potent stimulants of food intake in vertebrates, mammals and fish. However, the present knowledge about feeding behaviour in fish is still limited and based on studies in a few species. The Brazilian flounder Paralichthys orbignyanus is being considered for aquaculture, and it is important to understand the mechanisms regulating feeding in order to improve its performance in captivity. The objectives of this study were to clone NPY cDNA, evaluate the mRNA levels in different tissues of flounder, and also evaluate brain NPY expression to associate food intake with NPY expression levels. A 597 bp NPY cDNA was cloned from Brazilian flounder brain. NPY expression was detected in all the peripheral tissues analysed. No significant differences were observed in brain NPY gene expression over 24 h after food intake at a temperature of 15 ± 3°C. No correlation was observed among plasma glucose, total protein, cholesterol, triglycerides and NPY expression levels during this 24 h period. On the other hand, mRNA levels were increased after two weeks of fasting at elevated temperatures. Our results suggest that NPY mRNA levels in Brazilian flounder are affected by temperature.

SOCS1 and SOCS3 are the main negative modulators of the somatotrophic axis in liver of homozygous GH-transgenic zebrafish (Danio rerio).

Homozygote individuals (HO) of the GH-transgenic zebrafish lineage (F0104), despite expressing double the amount of growth hormone (GH) in relation to the hemizygote (HE) individuals, presented smaller growth in relation to the last, and similar to the non-transgenic (NT) group. Through the analysis of the expression of genes of the somatotrophic axis in the livers of HO and NT individuals, it was verified that GHR, JAK2 and STAT5.1 did not present significant differences among the analyzed genotypes (NT and HO). However, in the IGF-I gene expression, an accentuated decrease was observed in group HO (p<0.01), suggesting a resistance effect to excess GH. This resistance could be related to the insufficient amount of energy for supporting the accelerated metabolic demand caused by excess circulating GH. Analysis of the genes involved in the regulation of GH signalization by dephosphorylation (PTP-H1 and PTP-1B) did not show any significant alteration when comparing groups HO and NT. However, the analysis of the SOCS1 and SOCS3 genes showed an induction in homozygotes of 2.5 times (p<0.01) and 4.3 times (p<0.05), respectively, in relation to non-transgenics. The results of the present work demonstrate that, in homozygotes, GH signaling is reduced by the action of the SOCS1 and SOCS3 proteins.

Transcriptome of Atlantic Cod (Gadus morhua L.) Early Embryos from Farmed and Wild Broodstocks

Significant efforts have been made to elucidate factors affecting egg quality in fish. Recently, we have shown that eggs originating from wild broodstock (WB) of Atlantic cod (Gadus morhua L.) are of superior quality to those derived from farmed broodstock (FB), and this is associated with differences in the chemical composition of egg yolk. However, maternal transcripts, accumulated during oogenesis, have not been studied extensively in fish. The aim of the present study was to characterize putative maternal mRNA transcriptome in fertilized eggs of Atlantic cod and to compare transcript pools between WB and FB in order to investigate the relation between egg developmental potential and putative maternal mRNA deposits. We performed high-throughput 454 pyrosequencing. For each WB and FB group, five cDNA libraries were individually tagged and sequenced, resulting in 98,687 (WB) and 119,333 (FB) average reads per library. Sequencing reads were de novo assembled, annotated, and mapped. Out of 13,726 identified isotigs, 238 were differentially expressed between WB and FB, with 155 isotigs significantly upregulated in WB. The sequence reads were mapped to 11,340 different Atlantic cod transcripts and 158 sequences were differentially expressed between the 2 groups. Important transcripts involved in fructose metabolism, fatty acid metabolism, glycerophospholipid metabolism, and oxidative phosphorylation were differentially represented between the two broodstock groups, showing potential as biomarkers of egg quality in teleosts. Our findings contribute to the hypothesis that maternal mRNAs affect egg quality and, consequently, the early development of fish.

Growth hormone transgenesis affects osmoregulation and energy metabolism in zebrafish (Danio rerio)

Growth hormone (GH) transgenic fish are at a critical step for possible approval for commercialization. Since this hormone is related to salinity tolerance in fish, our main goal was to verify whether the osmoregulatory capacity of the stenohaline zebrafish (Danio rerio) would be modified by GH-transgenesis. For this, we transferred GH-transgenic zebrafish (T) from freshwater to 11 ppt salinity and analyzed survival as well as relative changes in gene expression. Results show an increased mortality in T versus non-transgenic (NT) fish, suggesting an impaired mechanism of osmotic acclimation in T. The salinity effect on expression of genes related to osmoregulation, the somatotropic axis and energy metabolism was evaluated in gills and liver of T and NT. Genes coding for Na+, K+-ATPase, H+-ATPase, plasma carbonic anhydrase and cytosolic carbonic anhydrase were up-regulated in gills of transgenics in freshwater. The growth hormone receptor gene was down-regulated in gills and liver of both NT and T exposed to 11 ppt salinity, while insulin-like growth factor-1 was down-regulated in liver of NT and in gills of T exposed to 11 ppt salinity. In transgenics, all osmoregulation-related genes and the citrate synthase gene were down-regulated in gills of fish exposed to 11 ppt salinity, while lactate dehydrogenase expression was up-regulated in liver. Na+, K+-ATPase activity was higher in gills of T exposed to 11 ppt salinity as well as the whole body content of Na+. Increased ATP content was observed in gills of both NT and T exposed to 11 ppt salinity, being statistically higher in T than NT. Taking altogether, these findings support the hypothesis that GH-transgenesis increases Na+ import capacity and energetic demand, promoting an unfavorable osmotic and energetic physiological status and making this transgenic fish intolerant of hyperosmotic environments.

Profiling of key apoptotic, stress, and immune-related transcripts during embryonic and postembryonic development of Atlantic cod (Gadus morhua L.)

We investigated the profiles of 25 genes involved in apoptosis (bcl-x2, casp3, casp8, ccar1, mcl1, and tpt1), immunity (bty, cathl, ifng, il1b, il6, il8, il10, lyzg, and tfa), oxidative stress (cat, gpx4, gsh-px, hsp70, hsp90a, and sod1), and stress axis (crh, pomc, grl1, and mlr) during Atlantic cod development and compared the mRNA transcript levels between samples from farmed (FB) and wild broodstock (WB) using real-time polymerase chain reaction. The suitability of nine endogenous housekeeping genes and an external standard (luciferase) as reference genes was also evaluated. The cycle threshold values of all housekeeping genes differed significantly throughout Atlantic cod development. Fertilization and hatching rates were significantly higher in WB group (95 ± 1.8% and 89 ± 2.8%, respectively) compared with FB (75 ± 3.4% and 66 ± 3.2%, respectively). Eleven target genes, namely, ccar1, casp3, bcl-x2, mcl-1, cat, gsh-px, hsp70, sod1, lyzg, il8, and grl were expressed in both groups at fertilization stage, indicating their maternal transfer. Among them, transcripts of gsh-px were more abundant in WB eggs, while the expression of hsp70 was significantly higher in FB eggs. In FB larvae, expression of cat, hsp70, hsp90a, pomc, mlr, grl1, bclx2, and il6 was significantly higher at hatching and the expression of cat, gpx4, casp3 and ccar1 was significantly higher at first feeding stages, than in WB group. These findings give an insight into the expressional changes in certain category of genes involved in the embryonic development of Atlantic cod, which may eventually determine the ultimate quality of the larvae.

GH overexpression causes muscle hypertrophy independent from local IGF-I in a zebrafish transgenic model

The aim of the present study was to analyse the morphology of white skeletal muscle in males and females from the GH-transgenic zebrafish (Danio rerio) lineage F0104, comparing the expression of genes related to the somatotrophic axis and myogenesis. Histological analysis demonstrated that transgenic fish presented enhanced muscle hypertrophy when compared to non-transgenic fish, with transgenic females being more hypertrophic than transgenic males. The expression of genes related to muscle growth revealed that transgenic hypertrophy is independent from local induction of insulin-like growth factor 1 gene (igf1). In addition, transgenic males exhibited significant induction of myogenin gene (myog) expression, indicating that myog may mediate hypertrophic growth in zebrafish males overexpressing GH. Induction of the α-actin gene (acta1) in males, independently from transgenesis, also was observed. There were no significant differences in total protein content from the muscle. Our results show that muscle hypertrophy is independent from muscle igf1, and is likely to be a direct effect of excess circulating GH and/or IGF1 in this transgenic zebrafish lineage.

Genotype-dependent gene expression profile of the antioxidant defense system (ADS) in the liver of a GH-transgenic zebrafish model.

The aim of this study was to evaluate the effects of growth hormone (GH) overexpression on the gene expression profile of multiple components of the antioxidant defense system (ADS) of different genotypes of a GH-transgenic zebrafish (Danio rerio) model. Several ADS-related genes were analyzed by semiquantitative reverse transcription–PCR in the liver of hemizygous (HE) and homozygous (HO) transgenic zebrafish. The results showed a significant reduction in the glutamate cysteine ligase catalytic subunit (GCLC) and the gene expression of two glutathione S-transferase (GST) isoforms and an increase in the glutathione reductase gene in the HO group compared to non-transgenic controls. The expression of the Cu, Zn-superoxide dismutase (SOD1) and catalase (CAT) genes was reduced in HO and HE groups, respectively. Among the ten genes analyzed, two were altered in HE transgenic zebrafish and five were altered in HO transgenic zebrafish. These findings indicate a genotype-dependent gene expression profile of the ADS-related genes in the liver of our GH-transgenic zebrafish model and are in agreement with the general effects of GH hypersecretion in the fish and mouse, which involves a reduction in the capability of the tissues to deal with oxidative stress situations. The GH-transgenic zebrafish model used here seems to be an interesting tool for analyzing the effect of different GH expression levels on physiological processes.

A comparative expression analysis of gene transcripts in brain tissue of non-transgenic and GH-transgenic zebrafish (Danio rerio) using a DDRT-PCR approach

The presence of higher level of exogenous growth hormone (GH) in transgenic animals could lead to several physiological alterations. A GH transgenic zebrafish (Danio rerio) line was compared to nontransgenic (NT) samples of the species through a DDRT-PCR approach, with the goal of identifying candidate differentially expressed transcripts in brain tissues that could be involved in GH overexpression. Densitometric analyses of two selected amplification products, p300 and ADCY2, pointed to a significant lower gene expression in the transgenic zebrafish (104.02 ± 57.71; 224.10 ± 91.73) when compared to NT samples (249.75 ± 30.08; 342.95 ± 65.19). The present data indicate that p300 and ADCY2 are involved in a regulation system for GH when high circulating levels of this hormone are found in zebrafishes.

Sperm-Mediated Gene Transfer: Concepts and Controversies

Description: Sperm-mediated gene transfer (SMGT) represents a novel set of technologies for animal (or in the future, human) genetic modification using the sperm as a vector, as opposed to more traditional established routes such as fertilized eggs or embryonic stem cells. Studies of sperm-mediated gene transfer (SMGT) indicate that sperm cells possess the ability to be utilized as carriers of exogenous genetic sequences, offering the potential of a novel cost-effective route for germline genetic modification. The fate of transgenes borne by sperm cells has been inconsistent, and analysis of offspring from SMGT experiments has shown a mixed picture in terms of genomic integration of the transgene, suggesting an episomal mode of inheritance. Various distinct steps in transgene uptake by the sperm cell have been described or proposed, including a model based upon endogenous reverse transcriptase activity. Although mature sperm cells are naturally protected against uptake of foreign nucleic acid molecules, certain environmental conditions, for example at key times within the reproductive tract, may reduce this protection, suggesting that SMGT may occasionally take place in nature. If correct, this carries profound implications for evolution and human genetic health. This e-book brings together theoretical and empirical reviews from experts in SMGT, providing comprehensive coverage of the major trends, developments and controversies in this novel field. This e-book is intended as a reference for professional researchers in the field of animal genetic modification (transgenesis) as well as teachers, scientists and physicians interested in medical genetics in general and gene therapy in particular.