The effect of melatonin implants during the seasonal anestrus on embryo production after superovulation in aged high-prolificacy Rasa Aragonesa ewes

The aim of this study was to assess the effect of melatonin implants administered in March on the ovarian cyclicity, ovulatory response and embryo production after repeated superovulation of selected high-prolificacy Rasa Aragonesa aged ewes. During the seasonal anestrus of two consecutive years, 113 superovulatory treatments have been performed. Ewes were treated (M) or not (C) with melatonin implants in March (day 0). All of them received intravaginal progestogen sponges on day 24 (recovery 1) and 80 (recovery 2) after melatonin implants insertion in year 1, and on day 28 and 77 in year 2. The intravaginal sponges were removed after 14 days. Superovulatory treatments consisted of eight doses in decreasing concentrations (2 mL x 2 and 1 mL x 6) of oFSH (Ovagen) administered twice daily starting 72 h before sponge removal. Seven days after the onset of estrus, embryos were recovered by laparotomy. Melatonin increased cyclicity only in recovery 2 year 2 (83% versus 42%; P < 0.05) but not in the other experimental periods. Among the 78% (88) ewes that ovulated and produced functional corpora lutea, melatonin implants tended to improve embryo viability in recovery 2 by increasing the number of blastocysts per superovulatory treatment (2.4 +/- 0.6 versus 1.1 +/- 0.4; P = 0.09), the rate of viability (67 +/- 9% versus 43 +/- 9%; P < 0.05), and freezability (55 +/- 9% versus 33 +/- 8%; P < 0.05). More specifically, melatonin induced a significant reduction of the number and rate of non-viable (degenerate and retarded) embryos in recovery 2 (0.4+/-0.1 embryos versus 1.3 +/- 0.3 embryos and 4 +/- 1% versus 22 +/- 6%, respectively; P < 0.05). Our results demonstrate that melatonin implants in March can improve at medium term (3 months after implantation) the viability of embryos collected from selected high-prolificacy Rasa Aragonesa aged ewes after superovulation.     

Digenic Inheritance in Cystinuria Mouse Model

Cystinuria is an aminoaciduria caused by mutations in the genes that encode the two subunits of the amino acid transport system b^0,+, responsible for the renal reabsorption of cystine and dibasic amino acids. The clinical symptoms of cystinuria relate to nephrolithiasis, due to the precipitation of cystine in urine. Mutations in SLC3A1, which codes for the heavy subunit rBAT, cause cystinuria type A, whereas mutations in SLC7A9, which encodes the light subunit b^0,+AT, cause cystinuria type B. By crossing Slc3a1^-/- with Slc7a9^-/- mice we generated a type AB cystinuria mouse model to test digenic inheritance of cystinuria. The 9 genotypes obtained have been analyzed at early (2- and 5-months) and late stage (8-months) of the disease. Monitoring the lithiasic phenotype by X-ray, urine amino acid content analysis and protein expression studies have shown that double heterozygous mice (Slc7a9^+/-Slc3a1^+/-) present lower expression of system b^0,+ and higher hyperexcretion of cystine than single heterozygotes (Slc7a9^+/-Slc3a1^+/+ and Slc7a9^+/+Slc3a1^+/-) and give rise to lithiasis in 4% of the mice, demonstrating that cystinuria has a digenic inheritance in this mouse model. Moreover in this study it has been demonstrated a genotype/phenotype correlation in type AB cystinuria mouse model providing new insights for further molecular and genetic studies of cystinuria patients.     

Substructural specificity and polyvalent carbohydrate recognition by the Entamoeba histolytica and rat hepatic N- acetylgalactosamine/galactose lectins

Both the Entamoeba histolytica lectin, a virulence factor for the causative agent of amebiasis, and the mammalian hepatic lectin bind to N-acetylgalactosamine (GalNAc) and galactose (Gal) nonreducing termini on oligosaccharides, with preference for GalNAc. Polyvalent GalNAc- derivatized neoglycoproteins have >1000-fold enhanced binding affinity for both lectins (Adler,P., Wood,S.J., Lee,Y.C., Lee,R.T., Petri,W.A.,Jr. and Schnaar,R.L.,1995, J. Biol. Chem ., 270, 5164-5171). Substructural specificity studies revealed that the 3-OH and 4-OH groups of GalNAc were required for binding to both lectins, whereas only the E.histolytica lectin required the 6-OH group. Whereas GalNAc binds with 4-fold lower affinity to the E.histolytica lectin than to the mammalian hepatic lectin, galactosamine and N-benzoyl galactosamine bind with higher affinity to the E. histolytica lectin. Therefore, a synthetic scheme for converting polyamine carriers to poly-N-acyl galactosamine derivatives (linked through the galactosamine primary amino group) was developed to test whether such ligands would bind the E.histolytica lectin with high specificity and high affinity. Contrary to expectations, polyvalent derivatives including GalN6lys5, GalN4desmosine, GalN4StarburstTMdendrimer, and GalN8StarburstTMdendrimer demonstrated highly enhanced binding to the mammalian hepatic lectin but little or no enhancement of binding to the E.histolytica lectin. We propose that the mammalian hepatic lectin binds with greatest affinity to GalNAc "miniclusters," which mimic branched termini of N-linked oligosaccharides, whereas the E.histolytica lectin binds most effectively to "maxiclusters," which may mimic more widely spaced GalNAc residues on intestinal mucins.      

Mitofusin-2 determines mitochondrial network architecture and mitochondrial metabolism. A novel regulatory mechanism altered in obesity

In many cells and specially in muscle, mitochondria form elongated filaments or a branched reticulum. We show that Mfn2 (mitofusin 2), a mitochondrial membrane protein that participates in mitochondrial fusion in mammalian cells, is induced during myogenesis and contributes to the maintenance and operation of the mitochondrial network. Repression of Mfn2 caused morphological and functional fragmentation of the mitochondrial network into independent clusters. Concomitantly, repression of Mfn2 reduced glucose oxidation, mitochondrial membrane potential, cell respiration, and mitochondrial proton leak. We also show that the Mfn2-dependent mechanism of mitochondrial control is disturbed in obesity by reduced Mfn2 expression. In all, our data indicate that Mfn2 expression is crucial in mitochondrial metabolism through the maintenance of the mitochondrial network architecture, and reduced Mfn2 expression may explain some of the metabolic alterations associated with obesity.     

Effects of ageing and exogenous melatonin on pituitary responsiveness to GnRH in ewes during anestrus and the reproductive season

The study examined the effect of melatonin implants on in vivo pituitary responsiveness to GnRH in control, fully productive (5.7+/-0.4 years old, n=17) and aged (10.7+/-0.3 years old, n=14) ovariectomized, estradiol-treated Rasa Aragonesa ewes. On 27 February, eight ewes in each age group received a single implant containing 18 mg melatonin. On 10 April, blood samples to be assayed for LH were collected at 10-min intervals over 4h (starting at 09:00 and 22:00 h). After samples 6 and 18 were collected, ewes received a single i.v. injection of GnRH (20 ng/kg liveweight). The pituitary response to GnRH was assessed using the difference between plasma LH concentrations before and after (highest value) each injection (DLH1, DLH2)), and the area under the LH response curve for 1h after each GnRH injection (AUC1, AUC2). On 23 September, the previously implanted ewes received a new melatonin implant and, on 17 November, all of the ewes were subjected to the same diurnal and nocturnal sampling protocols, again. Generally, non-implanted aged ewes exhibited a lower pituitary response to GnRH than did non-implanted control ewes, particularly in November and after the first injection (P<0.05 for DLH1 and AUC1 in both the diurnal and nocturnal tests). The response was significantly affected by the interaction of age and melatonin treatment, particularly in the diurnal tests (P<0.1 for DLH1 and AUC1, and P<0.05 for AUC2 in April; P<0.05 for DLH1, AUC1 and AUC2 in November), which indicated that exogenous melatonin increased LH levels after GnRH injections in aged ewes compared to non-implanted ewes, this effect being the opposite in control females. Thus, melatonin can restore in ewes the functionality of the neuroendocrine system, after it has been reduced by senescence.     

Tobamovirus evolution: gene overlaps, recombination, and taxonomic implications

Tobamoviruses, mostly isolated from solanaceous plants, may represent ancient virus lineages that have codiverged with their hosts. Recently completed nucleotide sequences of six nonsolanaceous tobamoviruses allowed assessment of the codivergence hypothesis and support a third subgroup within tobamoviruses. The genomic sequences of 12 tobamoviruses and the partial sequences of 11 others have been analyzed. Comparisons of the predicted protein sequences revealed three clusters of tobamoviruses, corresponding to those infecting solanaceous species (subgroup 1), those infecting cucurbits and legumes (subgroup 2), and those infecting crucifers. The orchid-infecting odontoglossum ringspot tobamovirus was associated with subgroup 1 genomes by its coat and movement protein sequences, but with the crucifer-pathogenic tobamoviruses by the remainder of its genome, suggesting that it is the progeny of a recombinant. For four of five genomic regions, subgroup 1 and 3 genomes were equidistant from a subgroup 2 genome chosen for comparison, suggesting uniform rates of evolution. A phylogenetic tree of plant families based on the tobamoviruses they harbor was congruent with that based on rubisco sequences but had a different root, suggesting that codivergence was tempered by rare events of viruses of one family colonizing another family. The proposed subgroup 3 viruses probably have an origin of virion assembly in the movement protein gene, a large (25-codon) overlap of movement and coat protein open reading frames, and a comparably shorter genome. Codon-position- dependent base compositions and codon prevalences suggested that the coat protein frame of the overlap region was ancestral. Bootstrapped parsimony analysis of the nucleotides in the overlap region and of the sequences translated from the -1 frame (the subgroup 3 movement protein frame) of this region produced trees inconsistent with those deduced from other regions. The results are consistent with a model in which a no or short overlap organization was ancestral. Despite encoding of subgroup 2 and 3 movement protein C-termini by nonhomologous nucleotides, weak similarities between their amino acid sequences suggested convergent sequence evolution.