Maternal ageing impairs mitochondrial DNA kinetics during early embryogenesis in mice.

STUDY QUESTION: Does ageing affect the kinetics of the mitochondrial pool during oogenesis and early embryogenesis? SUMMARY ANSWER: While we found no age-related change during oogenesis, the kinetics of mitochondrial DNA content and the expression of the factors involved in mitochondrial biogenesis appeared to be significantly altered during embryogenesis. WHAT IS KNOWN ALREADY: Oocyte mitochondria are necessary for embryonic development. The morphological and functional alterations of mitochondria, as well as the qualitative and quantitative mtDNA anomalies, observed during ovarian ageing may be responsible for the alteration of oocyte competence and embryonic development. STUDY DESIGN, SIZE, DURATION: The study, conducted from November 2016 to November 2017, used 40 mice aged 5-8 weeks and 45 mice aged 9-11 months (C57Bl6/CBA F(1)). A total of 488 immature oocytes, with a diameter ranging from 20 µm to more than 80 µm, were collected from ovaries, and 1088 mature oocytes or embryos at different developmental stages (two PN, one-cell, i.e. syngamy, two-cell, four-cell, eight-cell, morula and blastocyst) were obtained after ovarian stimulation and, for embryos, mating. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mitochondrial DNA was quantified by quantitative PCR. We used quantitative reverse transcriptase PCR (RT-PCR) (microfluidic method) to study the relative expression of three genes involved in the key steps of embryogenesis, i.e. embryonic genome activation (HSPA1) and differentiation (CDX2 and NANOG), two mtDNA genes (CYB and ND2) and five genes essential for mitochondrial biogenesis (PPARGC1A, NRF1, POLG, TFAM and PRKAA). The statistical analysis was based on mixed linear regression models applying a logistic link function (STATA v13.1 software), with values of P < 0.05 being considered significant. MAIN RESULTS AND THE ROLE OF CHANCE: During oogenesis, there was a significant increase in oocyte mtDNA content (P < 0.0001) without any difference between the two groups of mice (P = 0.73). During the first phase of embryogenesis, i.e. up to the two-cell stage, embryonic mtDNA decreased significantly in the aged mice (P < 0.0001), whereas it was stable for young mice (young/old difference P = 0.015). The second phase of embryogenesis, i.e. between the two-cell and eight-cell stages, was characterized by a decrease in embryonic mtDNA for young mice (P = 0.013) only (young/old difference P = 0.038). During the third phase, i.e. between the eight-cell and blastocyst stage, there was a significant increase in embryonic mtDNA content in young mice (P < 0.0001) but not found in aged mice (young/old difference P = 0.002). We also noted a faster expression of CDX2 and NANOG in the aged mice than in the young mice during the second (P = 0.007 and P = 0.02, respectively) and the third phase (P = 0.01 and P = 0.008, respectively) of embryogenesis. The expression of mitochondrial genes CYB and ND2 followed similar kinetics and was equivalent for both groups of mice, with a significant increase during the third phase (P < 0.01). Of the five genes involved in mitochondrial biogenesis, i.e. PPARGC1A, NRF1, POLG, TFAM and PRKAA, the expression of three genes decreased significantly during the first phase only in young mice (NRF1, P = 0.018; POLGA, P = 0.002; PRKAA, P = 0.010), with no subsequent difference compared to old mice. In conclusion, during early embryogenesis in the old mice, we suspect that the lack of a replicatory burst before the two-cell stage, associated with the early arrival at the minimum threshold value of mtDNA, together with the absence of an increase of mtDNA during the last phase, might potentially deregulate the key stages of early embryogenesis. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Because of the ethical impossibility of working on a human, this study was conducted only on a murine model. As superovulation was used, we cannot totally exclude that the differences observed were, at least partially, influenced by differences in ovarian response between young and old mice. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest a pathophysiological explanation for the link observed between mitochondria and the deterioration of oocyte quality and early embryonic development with age. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the University of Angers, France, by the French national research centres INSERM and the CNRS and, in part, by PHASE Division, INRA. There are no competing interests.

The mitochondrial DNA content of cumulus cells may help predict embryo implantation.

PURPOSE: The quantification of mtDNA in cumulus granulosa cells (CGCs) surrounding an oocyte has been positively linked with morphological embryonic quality. In the present study, we evaluated the link between the amount of mtDNA in CGCs surrounding an oocyte and the chances for the corresponding embryo of implanting and leading to an ongoing pregnancy. METHODS: This is an observational study, performed on 84 oocyte-cumulus-complexes (OCCs) having led to the replacement of an embryo in the maternal uterus, retrieved from 71 patients undergoing IVF with intracytoplasmic sperm. The OCCs were classified in two groups, one including 26 OCCs having led to an implanted embryo and the other including 58 OCCs having led to a non-implanted embryo. The average mtDNA content of CGCs was assessed by using a quantitative real-time PCR technique. RESULTS: Significantly higher mtDNA copy numbers in CGCs were associated with implanted embryos than with non-implanted embryos (mean 215 [sd 375] and 59 [sd 72], respectively; p < 104). Multivariate analysis, taking into account the women's age, the embryo quality, and the AMH level, suggests an independent relationship between the mtDNA content of CGCs and the potential of embryo implantation. CONCLUSION: During in vitro fertilization (IVF) procedures, the probability of the implantation of the embryo appears to be closely correlated to the mtDNA copy numbers in the CGCs. Our results highlight the interest of mtDNA quantification in GCGs as a biomarker of the potential of embryo implantation.

Mitochondrial complex I defect resulting from exercise-induced lower limb ischemia in patients with peripheral arterial disease.

This study aims to compare the structural and mitochondrial alterations between muscle segments affected by exercise-induced ischemia and segments of the same muscle without ischemia, in the same subject. In a prospective analysis, 34 patients presenting either peripheral arterial disease or chronic coronary syndrome without any evidence of peripheral arterial disease were eligible for inclusion based on findings indicating a need for either a femoro-popliteal bypass or a saphenous harvesting for coronary bypass. Before surgery, we assessed the level of exercise-induced ischemia in proximal and distal sections of the thigh by the measurement of transcutaneous oxygen pressure during an exercise treadmill test. Distal and proximal biopsies of the sartorius muscle were procured during vascular surgical procedures to assess mitochondrial function and morphometric parameters of the sartorius myofibers. Comparisons were made between the distal and proximal biopsies, with respect to these parameters. Thirteen of the study patients that initially presented with peripheral arterial disease had evidence of an isolated distal thigh exercise-induced ischemia, associated with a 35% decrease in the mitochondrial complex I enzymatic activity in the distal muscle biopsy. This defect was also associated with a decreased expression of the manganese superoxide dismutase enzyme and with alterations of the shapes of the myofibers. No functional or structural alterations were observed in the patients with coronary syndrome. We validated a specific model ischemia in peripheral arterial disease characterized by muscular alterations. This "Distal-Proximal-Sartorius Model" would be promising to explore the physiopathological consequences specific to chronic ischemia. NEW & NOTEWORTHY We compared proximal versus distal biopsies of the sartorius muscle in patients with superficial femoral artery stenosis or occlusion and proof of, distal only, regional blood flow impairment with exercise oximetry. We identified a decrease in the mitochondrial complex I enzymatic activity and antioxidant system impairment at the distal level only. We validate a model to explore the physiopathological consequences of chronic muscle ischemia.

Rabies surveillance in the Russian Federation.

Rabies is endemic to the Russian Federation. The disease incidence ranges between 2,000 and 4,000 cases annually. Between two and six cases in humans are also reported each year. Wild animals are the basic reservoir and vector of the virus, and the incidence of rabies in foxes and raccoon dogs amounts to 50% of the total number of disease cases. When outbreaks occur, the disease is also reported in domestic animals. To prevent the further spread of rabies, vaccination of domestic animals and oral immunisation of wildlife are practised. Unfortunately, vaccine coverage and disease prevention measures have not been sufficient to achieve a notable improvement in the rabies situation in the country.

La rage est présente à l'état endémique dans la Fédération de Russie. Son incidence varie de 2 000 à 4 000 cas par an. Deux à six cas de rage humaine sont également enregistrés chaque année. Les animaux sauvages constituent le principal réservoir et vecteur du virus, l'incidence de la rage chez le renard et le chien viverrin représentant 50 % du nombre total de cas. En cas de foyer, la maladie affecte également les animaux domestiques. La vaccination des animaux domestiques et l'immunisation orale de la faune sauvage sont les deux mesures appliquées pour empêcher la rage de se propager davantage. Malheureusement, la couverture vaccinale obtenue et les mesures de prévention appliquées n'ont pas suffi à améliorer significativement la situation de la rage.

La rabia es endémica en la Federación de Rusia, con una incidencia que va de los 2 000 a los 4 000 casos anuales. Cada año se notifican entre dos y seis casos en el ser humano. Los animales silvestres son el principal reservorio y vector del virus: la incidencia de la rabia en zorros y mapaches supone el 50% de la totalidad de los casos. Cuando estallan brotes también se registran casos en animales domésticos. Para impedir que la enfermedad se siga propagando se procede a la vacunación de los animales domésticos y a la inmunización oral de la fauna silvestre. Lamentablemente, la cobertura de vacunación y las medidas de prevención no han bastado para lograr una mejora sustancial de la situación de la rabia.

[Genetic characterization of the rabies virus field isolates detected in Russian Federation within the period 2008-2011].

Sixty-three gene N fragments of rabies virus field isolates detected within the period 2008-2011 in different regions of Russian Federation were sequenced. The comparison with previously tested isolates and strains has shown that newly isolated isolates can be placed into five previously described phylogenetic groups: Arctic group, Central Russian group, Eurasian group, Northern European group, and Caucasian group. The Arctic group isolates detected in Komi republic were identical to previously described rabies virus strain from Yakutia. This is the first reliable case of detecting Arctic group rabies virus in European part of Russia.

[Use of PCR and nucleotide sequence analysis of the VP7 gene for detection of bovine rotaviruses and identification of their G serotypes]. / Ispol'zovatie PTSR i sravnitel'nogo analiza nukleotidnoi posledovatel'nosti gena VP7 dlia vyiavlenia rotavirusov krupnogo rogatogo skota i opredeleniia ikh G-serotipov.

A Vp7 gene fragment PCR protocol was developed to detect the bovine rotaviruses and to identify their G serotypes. The most widespread bovine rotaviruses of G serotypes (G6, G8 and G10) can be distinguished on the basis of the PCR fragment size, while other G serotypes can be differentiated through a comparative analysis of the VP7 gene fragment nuclcotide sequence. Twenty-four bovine rotavirus field isolates were detected, and their G stereotypes were determined by using the method in question. Fourteen isolates were shown to be of G6 serotype; four of them were of G8, five--of G10, and one isolate was of G11 serotype. A possibility of detecting more than one isolate by this method was shown. Finally, a feasibility of using the method for searching for rotaviruses of new bovine rotavirus G serotypes and for rotaviruses, which do not belong to a so far described G serotypes, is discussed.

[Role of cholestanol in the pathogenesis of cholesterol cholelithiasis]. / Rol' kholestanola v patogeneze kholesterinovogo kholelitiaza.

The proportion of cholestanol/cholesterol in hepatic and cystic bile was evaluated using capillary gas-chromatography in subjects with disturbances in the biliary system (biliary dyskinesia, chronic acalculous cholecystitis, chronic calculous cholecystitis). The proportion was also measured in bile "paste" and cholesterol choleliths. With progression of cholesterol cholelithiasis, the proportion cholestanol/cholesterol increases, reaching its maximum in the stone the center of which accumulates crystals of cholestanol-cholesterol dihydrate. An original conception is proposed on pathogenesis of cholesterol cholelithiasis in man. It considers cholestanol as factor-reason underlying various pathological processes running in cholesterol cholelithiasis. Cholestanol is suggested to mark cholesterol cholelithiasis.

[The physicochemical evaluation of the bile using infrared spectroscopy]. / Fiziko-khimicheskaia otsenka zhelchi s pomoshch'iu infrakrasnoi spektroskopii.

Native bile, inspissated bile, and cholesterol choleliths were examined by infrared spectroscopy. Model compounds spectra were compared with native bile spectra. The spectra were divided into 3 groups: with acid fragments or cholesterol groups predominating and intermediate group. The peak of 3620 cm-1, absent in normal human bile and liable to augment if acid fragment level reduced was detected. The authors suggest a technique of infrared spectroscopy permitting early detection of disordered physicochemical characteristics of the bile in the course of cholesterol cholelithiasis development.

[Gas-chromatographic analysis of hypercholestanol in the bile and its role in the pathogenesis of cholelithiasis]. / Gazozhidkostno-khromatograficheskoe opredelenie giperkholestanol- kholii i ee znacheniev patogeneze kholelitiaza.

The cholestanol/cholesterol ratio in the bladder and liver bile was estimated by capillary gas-liquid chromatography in patients with dyskinesia of the gallbladder, chronic acalculous cholecystitis, and chronic calculous cholecystitis. Cholesterol biliary calculi and their surface and intermediate layers and the core were examined either. The authors came to a conclusion that cholestanol/cholesterol ratio in the bladder and liver bile reflected the progress of liver cholesterol metabolism starting from the early stages of cholelithiasis to the formation of cholesterol biliary calculi. Therefore hypercholestanolcholia should be considered as an indicator of the risk of cholesterol biliary calculi development.