Glycolytic pathway activity: effect on IVM and oxidative metabolism of bovine oocytes

The aim of the present study was to determine the effect of altering glycolytic pathway activity during bovine IVM on the meiotic maturation rate, oxidative activity, mitochondrial activity and the mitochondrial distribution within oocytes. Glycolytic activity was manipulated using two inhibitors (ATP, NaF) and a stimulator (AMP) of key enzymes of the pathway. Inhibition of glucose uptake, lactate production and meiotic maturation rates was observed when media were supplemented with ATP or NaF. The addition of AMP to the maturation medium had no effect on glucose uptake, lactate production or meiotic maturation. In the absence of gonadotrophin supplementation, AMP stimulated both glucose uptake and lactate production. However, AMP also decreased cytoplasmic maturation, as determined by early cleavage. During IVM, oocyte oxidative and mitochondrial activity was observed to increase at 15 and 22 h maturation. Inhibiting glycolysis with ATP or NaF led to a reduced oxidative and mitochondrial pattern compared with the respective control groups. Stimulation of the pathway with AMP increased oxidative and mitochondrial activity. A progressive mitochondrial migration to the central area was observed during maturation; oocytes treated with ATP, NaF or AMP showed limited migration. The present study reveals the effects of altering glycolytic pathway activity in cumulus–oocyte complexes, revealing the link between glycolysis of the cumulus–oocyte complex and the oxidative and mitochondrial activity of the oocyte.Cynthia Gutnisky, Sergio Morado, Gabriel C. Dalvit, Jeremy G. Thompson and Pablo D. Cetic

Genetic predisposition to hemophagocytic lymphohistiocytosis: report on 500 patients from the Italian registry

Background Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening disease affecting mostly children but also adults and characterized by hyperinflammatory features. A subset of patients, referred to as having familial hemophagocytic lymphohistiocytosis (FHL), have various underlying genetic abnormalities, the frequencies of which have not been systematically determined previously. Objective This work aims to further our understanding of the pathogenic bases of this rare condition based on an analysis of our 25 years of experience. Methods From our registry, we have analyzed a total of 500 unselected patients with HLH. Results Biallelic pathogenic mutations defining FHL were found in 171 (34%) patients; the proportion of FHL was much higher (64%) in patients given a diagnosis during the first year of life. Taken together, mutations of the genes PRF1 (FHL2) and UNC13D (FHL3) accounted for 70% of cases of FHL. Overall, a genetic diagnosis was possible in more than 90% of our patients with FHL. Perforin expression and the extent of degranulation have been more useful for diagnosing FHL than hemophagocytosis and the cytotoxicity assay. Of 281 (56%) patients classified as having "sporadic" HLH, 43 had monoallelic mutations in one of the FHL-defining genes. Given this gene dosage effect, FHL is not strictly recessive. Conclusion We suggest that the clinical syndrome HLH generally results from the combined effects of an exogenous trigger and genetic predisposition. Within this combination, different weights of exogenous and genetic factors account for the wide disease spectrum that ranges from HLH secondary to severe infection to FHL

Pentose phosphate pathway activity: effect on in vitro maturation and oxidative status of bovine oocytes

Abstract. The relationship between pentose phosphate pathway (PPP) activity in cumulus-oocyte complexes (COCs) and oxidative and mitochondrial activity in bovine oocytes was evaluated with the aim of analysing the impact of two inhibitors (NADPH and 6-aminonicotinamide (6-AN)) and a stimulator (NADP) of the key enzymes of the PPP on the maturation rate, oxidative and mitochondrial activity and the mitochondrial distribution in oocytes. The proportion of COCs with measurable PPP activity (assessed using brilliant cresyl blue staining), glucose uptake, lactate production and meiotic maturation rate diminished when 6-AN (0.1, 1, 5 and 10 mM for 22 h) was added to the maturation medium (P , 0.05). The addition of NADPH did not modify glucose uptake or lactate production, but reduced PPP activity in COCs and meiotic maturation rates (P , 0.05). The presence of NADP (0.0125, 0.125, 1.25 and 12.5 mM for 22 h of culture) in the maturation medium had no effect on PPP activity in COCs, glucose uptake, lactate production and meiotic maturation rate. However, in the absence of gonadotropin supplementation, NADP stimulated both glucose uptake and lactate production at 12.5 mM (the highest concentration tested; P , 0.05). NADP did not modify cleavage rate, but decreased blastocyst production (P , 0.05). During IVM, oocyte oxidative and mitochondrial activity was observed to increase at 15 and 22 h maturation, which was also related to progressive mitochondrial migration. Inhibiting the PPP with 6-AN or NADPH led to reduced oxidative and mitochondrial activity compared with the respective control groups and inhibition of mitochondrial migration (P , 0.05). Stimulation of the PPP with NADP increased oxidative and mitochondrial activity at 9 h maturation (P , 0.05) and delayed mitochondrial migration. The present study shows the significance of altering PPP activity during bovine oocyte IVM, revealing that there is a link between the activity of the PPP and the oxidative status of the oocyte

Clinical and genetic factors predicting Dravet syndrome in infants with SCN1A mutations

OBJECTIVE: To explore the prognostic value of initial clinical and mutational findings in infants with SCN1A mutations. METHODS: Combining sex, age/fever at first seizure, family history of epilepsy, EEG, and mutation type, we analyzed the accuracy of significant associations in predicting Dravet syndrome vs milder outcomes in 182 mutation carriers ascertained after seizure onset. To assess the diagnostic accuracy of all parameters, we calculated sensitivity, specificity, receiver operating characteristic (ROC) curves, diagnostic odds ratios, and positive and negative predictive values and the accuracy of combined information. We also included in the study demographic and mutational data of the healthy relatives of mutation carrier patients. RESULTS: Ninety-seven individuals (48.5%) had Dravet syndrome, 49 (23.8%) had generalized/genetic epilepsy with febrile seizures plus, 30 (14.8%) had febrile seizures, 6 (3.5%) had focal epilepsy, and 18 (8.9%) were healthy relatives. The association study indicated that age at first seizure and frameshift mutations were associated with Dravet syndrome. The risk of Dravet syndrome was 85% in the 0- to 6-month group, 51% in the 6- to 12-month range, and 0% after the 12th month. ROC analysis identified onset within the sixth month as the diagnostic cutoff for progression to Dravet syndrome (sensitivity = 83.3%, specificity = 76.6%). CONCLUSIONS: In individuals with SCN1A mutations, age at seizure onset appears to predict outcome better than mutation type. Because outcome is not predetermined by genetic factors only, early recognition and treatment that mitigates prolonged/repeated seizures in the first year of life might also limit the progression to epileptic encephalopathy

Mitochondria directly influence fertilisation outcome in the pig

The mitochondrion is explicitly involved in cytoplasmic regulation and is the cell's major generator of ATP. Our aim was to determine whether mitochondria alone could influence fertilisation outcome. In vitro, oocyte competence can be assessed through the presence of glucose-6-phosphate dehydrogenase (G6PD) as indicated by the dye, brilliant cresyl blue (BCB). Using porcine in vitro fertilisation (IVF), we have assessed oocyte maturation, cytoplasmic volume, fertilisation outcome, mitochondrial number as determined by mtDNA copy number, and whether mitochondria are uniformly distributed between blastomeres of each embryo. After staining with BCB, we observed a significant difference in cytoplasmic volume between BCB positive (BCB+) and BCB negative (BCB-) oocytes. There was also a significant difference in mtDNA copy number between fertilised and unfertilised oocytes and unequal mitochondrial segregation between blastomeres during early cleavage stages. Furthermore, we have supplemented BCB- oocytes with mitochondria from maternal relatives and observed a significant difference in fertilisation outcomes following both IVF and intracytoplasmic sperm injection (ICSI) between supplemented, sham-injected and non-treated BCB- oocytes. We have therefore demonstrated a relationship between oocyte maturity, cytoplasmic volume, and fertilisation outcome and mitochondrial content. These data suggest that mitochondrial number is important for fertilisation outcome and embryonic development. Furthermore, a mitochondrial pre-fertilisation threshold may ensure that, as mitochondria are diluted out during post-fertilisation cleavage, there are sufficient copies of mtDNA per blastomere to allow transmission of mtDNA to each cell of the post-implantation embryo after the initiation of mtDNA replication during the early postimplantation stages

Variations of the UNC13D gene in patients with autoimmune lymphoproliferative syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is caused by genetic defects decreasing Fas function and is characterized by lymphadenopathy/splenomegaly and expansion of CD4/CD8 double-negative T cells. This latter expansion is absent in the ALPS variant named Dianzani Autoimmune/lymphoproliferative Disease (DALD). In addition to the causative mutations, the genetic background influences ALPS and DALD development. We previously suggested a disease-modifying role for the perforin gene involved in familial hemophagocytic lymphohistiocytosis (FHL). The UNC13D gene codes for Munc13-4, which is involved in perforin secretion and FHL development, and thus, another candidate for a disease-modifying role in ALPS and DALD. In this work, we sequenced UNC13D in 21 ALPS and 20 DALD patients and compared these results with sequences obtained from 61 healthy subjects and 38 multiple sclerosis (MS) patients. We detected four rare missense variations in three heterozygous ALPS patients carrying p.Cys112Ser, p.Val781Ile, and a haplotype comprising both p.Ile848Leu and p.Ala995Pro. Transfection of the mutant cDNAs into HMC-1 cells showed that they decreased granule exocytosis, compared to the wild-type construct. An additional rare missense variation, p.Pro271Ser, was detected in a healthy subject, but this variation did not decrease Munc13-4 function. These data suggest that rare loss-of-function variations of UND13D are risk factors for ALPS development

Pentose phosphate pathway activity: effect on in vitro maturation and oxidative status of bovine oocytes

Published online: 17 July 2013The relationship between pentose phosphate pathway (PPP) activity in cumulus–oocyte complexes (COCs) and oxidative and mitochondrial activity in bovine oocytes was evaluated with the aim of analysing the impact of two inhibitors (NADPH and 6-aminonicotinamide (6-AN)) and a stimulator (NADP) of the key enzymes of the PPP on the maturation rate, oxidative and mitochondrial activity and the mitochondrial distribution in oocytes. The proportion of COCs with measurable PPP activity (assessed using brilliant cresyl blue staining), glucose uptake, lactate production and meiotic maturation rate diminished when 6-AN (0.1, 1, 5 and 10 mM for 22 h) was added to the maturation medium (P < 0.05). The addition of NADPH did not modify glucose uptake or lactate production, but reduced PPP activity in COCs and meiotic maturation rates (P < 0.05). The presence of NADP (0.0125, 0.125, 1.25 and 12.5 mM for 22 h of culture) in the maturation medium had no effect on PPP activity in COCs, glucose uptake, lactate production and meiotic maturation rate. However, in the absence of gonadotropin supplementation, NADP stimulated both glucose uptake and lactate production at 12.5 mM (the highest concentration tested; P < 0.05). NADP did not modify cleavage rate, but decreased blastocyst production (P < 0.05). During IVM, oocyte oxidative and mitochondrial activity was observed to increase at 15 and 22 h maturation, which was also related to progressive mitochondrial migration. Inhibiting the PPP with 6-AN or NADPH led to reduced oxidative and mitochondrial activity compared with the respective control groups and inhibition of mitochondrial migration (P < 0.05). Stimulation of the PPP with NADP increased oxidative and mitochondrial activity at 9 h maturation (P < 0.05) and delayed mitochondrial migration. The present study shows the significance of altering PPP activity during bovine oocyte IVM, revealing that there is a link between the activity of the PPP and the oxidative status of the oocyte.Cynthia Gutnisky, Gabriel C. Dalvit, Jeremy G. Thompson and Pablo D. Cetic