A variant associated to IGF-1 mRNA and protein expression in sheep.

The insulin-like growth factor-1 (IGF-1), a key hormone in muscle development was investigated for single-nucleotide polymorphisms (SNPs) upstream of the IGF-1 gene and their effects upon its cognate mRNA and hormone levels in sheep. A 70 d feeding trial was conducted with 22 F1 (Dorper × Pelibuey) lambs, individually allocated and fed a diet with a forage-to-concentrate ratio of 36:64 and 17% crude protein. Sequence analyses of 265 bp upstream the IGF-1 gene revealed the variant NC_040254.1:g.[184028491G > C;184028493G > A]. These SNPs generate alleles A and B, with frequencies of 0.66 and 0.34 in F1 lambs and of 0.73 and 0.27 in 81 pure Dorper lambs, respectively. Females were grouped by genotype AA, AB and BB (n = 3). IGF-1 hormone concentrations at 14, 42 and 70 d were higher (p < 0.05) in AA lambs compared to AB + BB lambs. The IGF-1 mRNA level was 2.6-fold higher in AA animals (n = 5, p < 0.05) than in AB + BB lambs (n = 7). A DNA binding site for the Inhibitor of Growth family member 4 (ING4) was found in allele B but not in allele A, which could explain the lower mRNA and hormone expression levels for AB + BB animals. The variant reported here appears to function as an eQTL with a negative effect on the level of IGF-1.

The glucose transporter 1 -GLUT1- from the white shrimp Litopenaeus vannamei is up-regulated during hypoxia.

During hypoxia the shrimp Litopenaeus vannamei accelerates anaerobic glycolysis to obtain energy; therefore, a correct supply of glucose to the cells is needed. Facilitated glucose transport across the cells is mediated by a group of membrane embedded integral proteins called GLUT; being GLUT1 the most ubiquitous form. In this work, we report the first cDNA nucleotide and deduced amino acid sequences of a glucose transporter 1 from L. vannamei. A 1619 bp sequence was obtained by RT-PCR and RACE approaches. The 5´ UTR is 161 bp and the poly A tail is exactly after the stop codon in the mRNA. The ORF is 1485 bp and codes for 485 amino acids. The deduced protein sequence has high identity to GLUT1 proteins from several species and contains all the main features of glucose transporter proteins, including twelve transmembrane domains, the conserved motives and amino acids involved in transport activity, ligands binding and membrane anchor. Therefore, we decided to name this sequence, glucose transporter 1 of L. vannamei (LvGLUT1). A partial gene sequence of 8.87 Kbp was also obtained; it contains the complete coding sequence divided in 10 exons. LvGlut1 expression was detected in hemocytes, hepatopancreas, intestine gills, muscle and pleopods. The higher relative expression was found in gills and the lower in hemocytes. This indicates that LvGlut1 is ubiquitously expressed but its levels are tissue-specific and upon short-term hypoxia, the GLUT1 transcripts increase 3.7-fold in hepatopancreas and gills. To our knowledge, this is the first evidence of expression of GLUT1 in crustaceans.

ADAMTS Proteases: Importance in Animal Reproduction.

Many reproductive physiological processes, such as folliculogenesis, ovulation, implantation, and fertilization, require the synthesis, remodeling, and degradation of the extracellular matrix (ECM). The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin Motifs) family genes code for key metalloproteinases in the remodeling process of different ECM. Several genes of this family encode for proteins with important functions in reproductive processes; in particular, ADAMTS1, 4, 5 and 9 are genes that are differentially expressed in cell types and the physiological stages of reproductive tissues. ADAMTS enzymes degrade proteoglycans in the ECM of the follicles so that the oocytes can be released and regulate follicle development during folliculogenesis, favoring the action of essential growth factors, such as FGF-2, FGF-7 and GDF-9. The transcriptional regulation of ADAMTS1 and 9 in preovulatory follicles occurs because of the gonadotropin surge in preovulatory follicles, via the progesterone/progesterone receptor complex. In addition, in the case of ADAMTS1, pathways involving protein kinase A (PKA), extracellular signal regulated protein kinase (ERK1/2) and the epidermal growth factor receptor (EGFR) might contribute to ECM regulation. Different Omic studies indicate the importance of genes of the ADAMTS family from a reproductive aspect. ADAMTS genes could serve as biomarkers for genetic improvement and contribute to enhance fertility and animal reproduction; however, more research related to these genes, the synthesis of proteins encoded by these genes, and regulation in farm animals is needed.

The glyceraldehyde-3-phosphate dehydrogenase of the shrimp Litopenaeus vannamei: Molecular cloning, characterization and expression during hypoxia.

Some marine crustaceans like the white shrimp Litopenaeus vannamei are tolerant to environmental hypoxia. Under oxygen deprivation, shrimp tissues obtain energy by enhancing anaerobic glycolysis. In mammals, hypoxia increases the expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which has been shown a "moonlighting" role in cells. However, the effect of hypoxia on the GAPDH expression has not been studied in crustaceans. In the present work, we obtained a 2744 bp gene sequence with a 999 bp ORF split by a single intron. The deduced protein is 332 amino acids and corresponds to the L. vannamei GAPDH (LvGAPDH), which is highly similar in sequence and structure to other animal GAPDHs. During hypoxia, LvGAPDH expression is significantly induced in gills but not in hepatopancreas, suggesting that it may play a role in the molecular and cellular response of shrimp to hypoxia.

Hypoxia drives apoptosis independently of p53 and metallothionein transcript levels in hemocytes of the whiteleg shrimp Litopenaeus vannamei.

The cellular mechanisms used by the shrimp Litopenaeus vannamei to respond to hypoxia have been studied from the energetic metabolism and antioxidant angles. We herein investigated the participation of p53 and metallothionein (MT) in the apoptotic process in response to hypoxia in shrimp hemocytes. The Lvp53 or LvMT genes were efficiently silenced by injection of double stranded RNA for p53 or MT. The effects of silencing on apoptosis were measured as caspase-3 activity and flow cytometry in hemocytes after 24 and 48 h of hypoxia (1.5 mg DO L(-1)). Hemocytes from unsilenced animals had significantly higher apoptosis levels upon both times of hypoxia. The apoptotic levels were diminished but not suppressed in dsp53-silenced but not dsMT-silenced hemocytes after 24 h of hypoxia, indicating a contribution of Lvp53 to apoptosis. Apoptosis in normoxia was significantly higher in dsp53-and dsMT-silenced animals compared to the unsilenced controls, pointing to a possible cytoprotective role of LvMT and Lvp53 during the basal apoptotic program in normoxia. Overall, these results indicate that hypoxia augments apoptosis in shrimp hemocytes and high mRNA levels of Lvp53 and LvMT are not necessary for this response.

A novel functional glucose transporter in the white shrimp Litopenaeus vannamei -LvGLUT2- is up-regulated during hypoxia in hepatopancreas.

In hypoxia conditions, the white shrimp Litopenaeus vannamei shifts its energetic metabolism from aerobic to anaerobic, requiring more glucose uptake into the cells by GLUT proteins. We here report a novel glucose transporter in shrimp. The Lvglut2 cDNA is 2473 bp-long containing an ORF of 1458 bp encoding 486 amino acid residues. The deduced protein has the features of a facilitative sugar transporter. The Lvglut2 gene product tagged with GFP was expressed in the cell membrane of Xenopus oocytes. In the same expression system, untagged LvGLUT2 resulted to be a bidirectional glucose transporter that functions moving glucose down its concentration gradient in and out of the cell. Lvglut2 mRNA is expressed in hepatopancreas while in muscle and gills it was not detected. Hypoxia up-regulates the expression of Lvglut2 transcripts in hepatopancreas. These results provide a better understanding of facilitative glucose transporters and gene regulation during hypoxia in crustaceans.

Expression of fructose 1,6-bisphosphatase and phosphofructokinase is induced in hepatopancreas of the white shrimp Litopenaeus vannamei by hypoxia.

Marine organisms are exposed to hypoxia in natural ecosystems and during farming. In these circumstances marine shrimp survive and synthesize ATP by anaerobic metabolism. Phosphofructokinase (PFK) and fructose 1,6-bisphosphatase (FBP) are key enzymes in carbohydrate metabolism. Here we report the cDNA of FBP from the shrimp Litopenaeus vannamei hepatopancreas and expression of PFK and FBP under normoxia and hypoxia. Hypoxia induces PFK and FBP expression in hepatopancreas but not in gills and muscle. Induction in hepatopancreas of the glycolytic and gluconeogenic key enzymes, PFK and FBP, suggests that PFK could be a key factor for increasing anaerobic rate, while FBP is probably involved in the activation of gluconeogenesis or the pentose-phosphates pathway during hypoxia in the highly active metabolism of hepatopancreas.

The metallothionein gene from the white shrimp Litopenaeus vannamei: characterization and expression in response to hypoxia.

Aquatic animals encounter variation in oxygen tension that leads to the accumulation of reactive oxygen species (ROS) that can harm the organisms. Under these circumstances some organisms have evolved to tolerate hypoxia. In mammals, metallothioneins (MTs) protect against hypoxia-generated ROS. Here we report the MT gene from the shrimp Litopenaeus vannamei (LvMT). LvMT is differentially expressed in hemocytes, intestine, gills, pleopods, heart, hepatopancreas and muscle, with the highest levels in hepatopancreas and heart. LvMT mRNA increases during hypoxia in hepatopancreas and gills after 3 h at 1.5 mg L(-1) dissolved oxygen (DO). This gene structure resembles the homologs from invertebrates and vertebrates possessing three exons, two introns and response elements for metal response transcription factor 1 (MTF-1), hypoxia-inducible factor 1 (HIF-1) and p53 in the promoter region. During hypoxia, HIF-1/MTF-1 might participate inducing MT to contribute towards the tolerance to ROS toxicity. MT importance in aquatic organisms may include also ROS-detoxifying processes.