86 Effect of invitro culture conditions on mitochondria functions in mouse embryos

2020 ◽  
Vol 32 (2) ◽  
pp. 169
Author(s):  
M. Czernik ◽  
D. Winiarczyk ◽  
S. Sampino ◽  
P. Greda ◽  
J. A. Modlinski ◽  
...  
Keyword(s):  
Embryo Development ◽  
Copy Number ◽  
Energy Demand ◽  
Mitochondrial Membrane ◽  
Mitochondrial Dynamics ◽  
Culture Conditions ◽  
Mouse Embryos ◽  
Mtdna Copy Number ◽  
Mitochondrial Functions ◽  
Mitochondria Functions

Mitochondria provide the energy for oocyte maturation, fertilisation, and embryo formation via oxidative phosphorylation. Consequently, any adverse influence on mitochondrial function may negatively affect the development of pre-implantation embryos especially because there is no mitochondrial DNA (mtDNA) replication until post-implantation. Studies in the field of mitochondrial dynamics have identified an intriguing link between energy demand/supply balance and mitochondrial architecture, which may suggest that inappropriate culture conditions may inhibit mitochondrial functions, which may negatively affect embryo development. We wanted to check whether invitro culture (IVC) conditions of mouse embryos affect mitochondrial functionality. The IVC as well as naturally matted (NM) mouse embryos at the 2-cell and blastocyst stage were subjected to mitochondrial analysis (distribution, organisation, and mitochondrial membrane potential), and expression of mRNA and proteins involved in regulation of mitochondria functions, as well as number of mtDNA copies, were evaluated. Significance level was set at 0.05. We observed that the mitochondria in 2-cell IVC embryos were less numerous and localised mainly in the pericortical region of the cytoplasm, whereas mitochondria in NM embryos were numerous and homogeneously distributed in both blastomeres. Drastic differences were observed in blastocysts. Mitochondria in the IVC group were fragmented, rounded, and aggregated mainly in the perinuclear region of the cells, whereas mitochondria of NM blastocysts were numerous and created an elongated mitochondrial network along the cells. Time-lapse analysis showed reduced mitochondrial and mitochondrial membrane activity in IVC blastocysts. Moreover, our results indicate the IVC group had reduced mRNA expression of mitofusin 1, mitofusin 2, and optic atrophy 1 responsible for mitochondrial fusion. Additionally, mtDNA copy number for IVC blastocysts (398 887.45±30 608.65) was significantly lower than that of NM blastocysts (593 367.12±66 540.32; P<0.02). Furthermore, no significant differences were found in mtDNA copy number of IVC 2-cell embryos when compared with NM embryos. The results obtained clearly showed that IVC conditions affect proper mitochondrial functionality and hence embryo development.

scholarly journals Variability in mitochondrial import, mitochondrial health and mtDNA copy number using Type II and Type V CRISPR effectors

2020 ◽  
Author(s):  
Zuriñe Antón ◽  
Grace Mullally ◽  
Holly Ford ◽  
Marc W. van der Kamp ◽  
Mark D. Szczelkun ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Copy Number ◽  
Protein Targeting ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Protein ◽  
Mitochondrial Diseases ◽  
Basic Research ◽  
Strategy Development ◽  
Mtdna Copy Number ◽  
Mitochondrial Import

ABSTRACTCurrent methodologies for targeting the mitochondrial genome for basic research and/or therapeutic strategy development in mitochondrial diseases are restricted by practical limitations and technical inflexibility. The development of a functional molecular toolbox for CRISPR-mediated mitochondrial genome editing is therefore desirable, as this could enable precise targeting of mtDNA haplotypes using the precision and tuneability of CRISPR enzymes; however, published reports of “MitoCRISPR” systems have, to date, lacked reproducibility and independent corroboration. Here, we have explored the requirements for a functional MitoCRISPR system in human cells by engineering several versions of CRISPR nucleases, including the use of alternative mitochondrial protein targeting sequences and smaller paralogues, and the application of gRNA modifications that reportedly induce mitochondrial import. We demonstrate varied mitochondrial targeting efficiencies and influences on mitochondrial dynamics/function of different CRISPR nucleases, with Lachnospiraceae bacterium ND2006 (Lb) Cas12a being better targeted and tolerated than Cas9 variants. We also provide evidence of Cas9 gRNA association with mitochondria in HeLa cells and isolated yeast mitochondria, even in the absence of a targeting RNA aptamer. Finally, we present evidence linking mitochondrial-targeted LbCas12a/crRNA with increased mtDNA copy number dependent upon DNA binding and cleavage activity. We discuss reproducibility issues and the future steps necessary if MitoCRISPR is to be realised.

Mitochondrial DNA replication is initiated at blastocyst formation in equine embryos

2019 ◽  
Vol 31 (3) ◽  
pp. 570 ◽  
Author(s):  
W. Karin Hendriks ◽  
Silvia Colleoni ◽  
Cesare Galli ◽  
Damien B. B. P. Paris ◽  
Ben Colenbrander ◽  
...  
Keyword(s):  
Dna Replication ◽  
Copy Number ◽  
Oocyte Quality ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Mtdna Copy Number ◽  
Blastocyst Development ◽  
Mt Dna ◽  
Mitochondrial Transcription Factor ◽  
Mitochondrial Number

Intracytoplasmic sperm injection is the technique of choice for equine IVF and, in a research setting, 18–36% of injected oocytes develop to blastocysts. However, blastocyst development in clinical programs is lower, presumably due to a combination of variable oocyte quality (e.g. from old mares), suboptimal culture conditions and marginal fertility of some stallions. Furthermore, mitochondrial constitution appears to be critical to developmental competence, and both maternal aging and invitro embryo production (IVEP) negatively affect mitochondrial number and function in murine and bovine embryos. The present study examined the onset of mitochondrial (mt) DNA replication in equine embryos and investigated whether IVEP affects the timing of this important event, or the expression of genes required for mtDNA replication (i.e. mitochondrial transcription factor (TFAM), mtDNA polymerase γ subunit B (mtPOLB) and single-stranded DNA binding protein (SSB)). We also investigated whether developmental arrest was associated with low mtDNA copy number. mtDNA copy number increased (P<0.01) between the early and expanded blastocyst stages both invivo and invitro, whereas the mtDNA:total DNA ratio was higher in invitro-produced embryos (P=0.041). Mitochondrial replication was preceded by an increase in TFAM but, unexpectedly, not mtPOLB or SSB expression. There was no association between embryonic arrest and lower mtDNA copy numbers.

scholarly journals Mitochondrial import, health and mtDNA copy number variability seen when using type II and type V CRISPR effectors

2020 ◽  
Vol 133 (18) ◽  
pp. jcs248468 ◽  
Author(s):  
Zuriñe Antón ◽  
Grace Mullally ◽  
Holly C. Ford ◽  
Marc W. van der Kamp ◽  
Mark D. Szczelkun ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Copy Number ◽  
Protein Targeting ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Protein ◽  
Mitochondrial Diseases ◽  
Mtdna Copy Number ◽  
Data Link ◽  
Mitochondrial Import ◽  
Type V

ABSTRACTCurrent methodologies for targeting the mitochondrial genome for research and/or therapy development in mitochondrial diseases are restricted by practical limitations and technical inflexibility. A molecular toolbox for CRISPR-mediated mitochondrial genome editing is desirable, as this could enable targeting of mtDNA haplotypes using the precision and tuneability of CRISPR enzymes. Such ‘MitoCRISPR’ systems described to date lack reproducibility and independent corroboration. We have explored the requirements for MitoCRISPR in human cells by CRISPR nuclease engineering, including the use of alternative mitochondrial protein targeting sequences and smaller paralogues, and the application of guide (g)RNA modifications for mitochondrial import. We demonstrate varied mitochondrial targeting efficiencies and effects on mitochondrial dynamics/function of different CRISPR nucleases, with Lachnospiraceae bacterium ND2006 (Lb) Cas12a being better targeted and tolerated than Cas9 variants. We also provide evidence of Cas9 gRNA association with mitochondria in HeLa cells and isolated yeast mitochondria, even in the absence of a targeting RNA aptamer. Our data link mitochondrial-targeted LbCas12a/crRNA with increased mtDNA copy number dependent upon DNA binding and cleavage activity. We discuss reproducibility issues and the future steps necessary for MitoCRISPR.

scholarly journals Screening for Mitochondrial tRNA Mutations in 318 Patients with Dilated Cardiomyopathy

2022 ◽  
Author(s):  
Yujuan Qi ◽  
Zhenhua Wu ◽  
Yaobang Bai ◽  
Yan Jiao ◽  
Peijun Li
Keyword(s):  
Dilated Cardiomyopathy ◽  
Mitochondrial Dysfunction ◽  
Copy Number ◽  
Mutational Analysis ◽  
Unknown Etiology ◽  
Trna Genes ◽  
Mtdna Copy Number ◽  
Mitochondrial Trna ◽  
Mitochondrial Functions ◽  
And Gender

Objectives: Dilated cardiomyopathy (DCM) is a complex cardiovascular disease with unknown etiology. Although nuclear genes play active roles in DCM, mitochondrial dysfunction was believed to be involved in the pathogenesis of DCM. The objective of this study is to analysis the association between mitochondrial tRNA (mt-tRNA) mutations and DCM. Material and Methods: We performed a mutational analysis of mt-tRNA genes in a cohort of 318 patients with DCM and 200 age- and gender-matched control subjects. To further assess their pathogenicity, phylogenetic analysis and mitochondrial functions including mtDNA copy number, ATP and ROS were analyzed. Results: 7 possible pathogenic mutations: MT-TL1 3302A>G, MT-TI 4295A>G, MT-TM 4435A>G, MT-TA 5655T>C, MT-TH 12201T>C, MT-TE 14692A>G and MT-TT 15927G>A were identified in DCM group but absent in controls. These mutations occurred at extremely conserved nucleotides of corresponding tRNAs, and led to the failure in tRNAs metabolism. Moreover, a significant reduction in ATP and mtDNA copy number, whereas a markedly increased in ROS level were observed in polymononuclear leukocytes (PMNs) derived from the DCM patients carrying these mt-tRNA mutations, suggesting that these mutations may cause mitochondrial dysfunction that was responsible for DCM. Conclusions: Our data indicated that mt-tRNA mutations may be the molecular basis for DCM, which shaded novel insight into the pathophysiology of DCM that was manifestated by mitochondrial dysfunction.

6 THE EFFECTS OF DEPLETING DONOR CELL MITOCHONDRIAL DNA ON CATTLE EMBRYOS DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER

2016 ◽  
Vol 28 (2) ◽  
pp. 132 ◽  
Author(s):  
K. Srirattana ◽  
J. C. St. John
Keyword(s):  
Mitochondrial Dna ◽  
Somatic Cell ◽  
Embryo Development ◽  
Copy Number ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Donor Cell ◽  
Mtdna Copy Number ◽  
Donor Cells

Although somatic cell nuclear transfer (SCNT) is a valuable tool for producing animals for agricultural and research purposes, the resultant mixing of mitochondrial DNA (mtDNA) from the donor cell and recipient oocyte (heteroplasmy) affects embryo development and offspring survival and health. The aim of this study was to determine the effects of depleting donor cells of their mtDNA before SCNT on embryo development. mtDNA was depleted from cattle fibroblasts using 2′,3′-dideoxycytidine. mtDNA copy number in cells depleted for 30 days (0.85 ± 0.05) was significantly decreased when compared with nondepleted cells (150.12 ± 29.90; P < 0.0001, ANOVA). Moreover, mtDNA copy number in depleted cells could not be replenished after depletion for 30 days. Depleted cells and nondepleted cells were used as donor cells for SCNT. Somatic cell nuclear transfer embryos were produced by electrofusion of a single donor cell with an enucleated cow oocyte. Reconstructed oocytes were chemically activated and cultured for 7 days (nontreated embryos). Another cohort of embryos was treated with Trichostatin A (TSA), to enhance reprogramming, by activating reconstructed oocytes and culturing them in the presence of 50 nM TSA for up to 10 h. The embryos were then cultured in the absence of TSA. In nontreated groups, the fusion rates of depleted cells (78.0 ± 0.8%) were significantly lower than those of nondepleted cells (92.1 ± 1.4%; P < 0.05). No positive effect on fusion rates was found after TSA treatment. The blastocyst rate for SCNT embryos derived from depleted cells (18.7 ± 4.9%) was significantly lower than the nondepleted group (32.5 ± 3.1%; P < 0.05). Trichostatin A treatment increased blastocyst rates for SCNT embryos derived from depleted cells (32.5 ± 5.3%) to levels equivalent to those of nondepleted cells but did not have any beneficial effect on SCNT embryos derived from nondepleted cells. We have analysed blastocysts for the presence of donor cell mtDNA by high resolution melting analysis. Four out of 10 SCNT blastocysts derived from nondepleted cells were heteroplasmic, whereas others had no donor cell mtDNA. However, all 10 analysed SCNT blastocysts derived from depleted cells were homoplasmic as they harboured only oocyte mtDNA. From RNA sequencing results, TSA treatment of SCNT blastocysts derived from depleted cells increased the expression of key developmental transcription regulators and decreased expression of the mtDNA-specific replication factors, which is essential for embryo development. In conclusion, homoplasmic SCNT embryos were successfully produced by using mtDNA depleted donor cells. Trichostatin A treatment enhanced nuclear reprogramming efficiency in SCNT embryos derived from depleted cells. This work was supported by MitoStock Pty. Ltd., Australia.

scholarly journals What Drives Embryo Development? Chromosomal Normality or Mitochondria?

2017 ◽  
Vol 2017 ◽  
pp. 1-4
Author(s):  
A. Bayram ◽  
I. Elkhatib ◽  
A. Arnanz ◽  
A. Linan ◽  
F. Ruiz ◽  
...  
Keyword(s):  
Embryo Development ◽  
Copy Number ◽  
Preimplantation Genetic Screening ◽  
Fertilization Rate ◽  
Normal Embryo ◽  
Mtdna Copy Number ◽  
Fertility Clinic ◽  
Next Generation Sequencing Ngs ◽  
Further Development ◽  
Generation Sequencing

Objective. To report the arrest of euploid embryos with high mtDNA content. Design. A report of 2 cases. Setting. Private fertility clinic. Patients. 2 patients, 45 and 40 years old undergoing IVF treatment. Interventions. Mature oocytes were collected and vitrified from two ovarian stimulations. Postthaw, survived mature oocytes underwent fertilization by intracytoplasmic sperm injection (ICSI). Preimplantation genetic screening (PGS) and mitochondrial DNA (mtDNA) copy number were done using next generation sequencing (NGS). The only normal embryo among the all-biopsied embryos had the highest “Mitoscore” value and was the only arrested embryo in both cases. Therefore, the embryo transfer was cancelled. Main Outcome Measures. Postthaw survival and fertilization rate, embryo euploidy, mtDNA copy number, and embryo development. Results. In both patients, after PGS only 1 embryo was euploid. Both embryos had the highest mtDNA copy number from all tested embryos and both embryos were arrested on further development. Conclusions. These cases clearly demonstrate the lack of correlation between mtDNA value (Mitoscore) and chromosomal status of embryo.

scholarly journals Sperm of patients with severe asthenozoospermia show biochemical, molecular and genomic alterations

Reproduction ◽  
2016 ◽  
Vol 152 (6) ◽  
pp. 695-704 ◽  
Author(s):  
Oriana Bonanno ◽  
Giulietta Romeo ◽  
Paola Asero ◽  
Franca Maria Pezzino ◽  
Roberto Castiglione ◽  
...  
Keyword(s):  
Copy Number ◽  
Mitochondrial Membrane ◽  
Nuclear Dna ◽  
Pathological Condition ◽  
Ros Production ◽  
Oxygen Species ◽  
Mtdna Copy Number ◽  
Sperm Abnormalities ◽  
Fertilizing Ability ◽  
Nuclear Dna Fragmentation

The multifactorial pathological condition, that is, severe low sperm motility is a frequent cause of infertility. However, mechanisms underlying the development of this condition are not completely understood. Single abnormalities have been reported in sperm of patients with asthenozoospermia. In this study, we characterized, in 22 normozoospermic men and in 37 patients with asthenozoospermia, biochemical, molecular and genomic abnormalities that frequently occur in sperm of patients with asthenozoospermia. We evaluated a panel of sperm biomarkers that may affect the motility and fertilizing ability of sperm of patients with severe asthenozoospermia. Since reactive oxygen species (ROS) production is involved in the pathogenesis of such sperm abnormalities, we determined the association between ROS production and sperm abnormalities. High percentage of patients with severe asthenozoospermia showed increased basal and stimulated ROS production. Moreover, these patients showed increased mitochondrial DNA (mtDNA) copy number but decreased mtDNA integrity and they were associated with elevated ROS levels. Furthermore, mitochondrial membrane potential was also significantly decreased and again associated with high ROS production in these patients. However, the rate of nuclear DNA fragmentation was increased only in less than one-fifth of these patients. An important cohort of these patients showed multiple identical biochemical, molecular and genomic abnormalities, which are typical manifestations of oxidative stress. The most frequent association was found in patients with high ROS levels, increased mtDNA copy number and decreased integrity, and low MMP. A smaller cohort of the aforementioned patients also showed nDNA fragmentation. Therefore, patients with asthezoospermia likely present reduced fertilizing potential because of such composed abnormalities.

191 SUPPLEMENTATION OF MATURATION MEDIUM WITH FOLLICULAR FLUID, EPIDERMAL GROWTH FACTOR AND NEUREGULIN AFFECTS MITOCHONDRIAL DNA REPLICATION, OOCYTE MATURATION AND EMBRYO DEVELOPMENT IN PIGS

2012 ◽  
Vol 24 (1) ◽  
pp. 208
Author(s):  
J. Mao ◽  
K. M. Whitworth ◽  
L. D. Spate ◽  
E. M. Walters ◽  
J. Zhao ◽  
...  
Keyword(s):  
Growth Factor ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Follicular Fluid ◽  
Copy Number ◽  
In Vitro Maturation ◽  
Meiotic Maturation ◽  
Mtdna Copy Number ◽  
Maturation Medium

Mitochondria supply the majority of ATP in a cell. Mitochondrial DNA (mtDNA) copy number in oocytes might be used as a marker of viability and might be a key determinant of pre-implantation embryo development. However, little is known about mtDNA copy number changes during porcine oocyte maturation and its regulation by extracellular growth factors. The objectives of the current study were to determine the effects of supplementation of in vitro maturation medium with porcine follicular fluid (pFF; 0, 10, 20 and 30%), epidermal growth factor (EGF; 10 ng mL–1), neuregulin 1 (NRG; 20 ng mL–1) and NRG + IGF1 (insulin-like growth factor-1; 100 ng mL–1 + NRG, 20 ng mL–1) during in vitro maturation on mtDNA copy number, oocyte meiotic maturation and subsequent embryo development after parthenogenic activation. Follicular fluid used for the pFF supplementation experiment was prepared from medium-sized (3–6 mm in diameter) healthy follicles. Cumulus–oocyte complexes (COCs) were collected from antral follicles (3–6 mm in diameter), cultured in LH- and FSH-containing maturation medium for 22 h at 38.5°C, transferred into basic maturation medium without FSH and LH and cultured for another 22 h. The basic maturation medium was TCM-199 supplemented with 0.1% polyvinylalcohol (w/v), 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 10 μg mL–1 of gentamicin, 0.57 mM cysteine and without or with different growth factors depending on the experimental design. In total, 177 germinal vesicle (GV) oocytes and 3837 MII oocytes were used for this study. All data were analyzed by the general linear model (GLM) procedure of SAS software (V9.2). The mtDNA copy number in oocytes increased (P < 0.05) from GV to MII stage oocytes (MII oocytes from all treatment groups pooled). Supplementation of IVM media with 10% pFF decreased mtDNA copy number (P < 0.05), whereas 20 and 30% pFF had no major effect on mtDNA copy number, resulting in a quadratic correlation between percentage of pFF and mtDNA copy number. There was a negative linear correlation between percentage of pFF and oocyte meiotic maturation, with a higher percentage of pFF inhibiting meiotic maturation (73.2 ± 5.2, 71.9 ± 4.8, 64.1 ± 8.5 and 65.8 ± 6.4% for 0, 10, 20 and 30% pFF groups, respectively). The mtDNA copy numbers in EGF and NRG-treated MII oocytes were significantly higher than those in GV oocytes, whereas the control was not different (EGF, 237 042.6 ± 22 198.2; NRG, 281 293.4 ± 22 893.5; and control, 231 856.8 ± 21 883.5 in MII oocytes vs 192 288.7 ± 21 675.4 in GV oocytes). The EGF, NRG and NRG+IGF1 treatments enhanced oocyte maturation as well. There was no difference in Day-7 blastocyst formation between EGF, NRG+IGF1 and the control, whereas the NRG treatment enhanced blastocyst formation as compared to the control (23.8 ± 2.4 vs 15.1 ± 2.1%; P < 0.05). This study demonstrated that there was an increase in mtDNA copy number during in vitro maturation. The EGF and NRG treatments stimulated mitochondria biogenesis, which may provide new means to increase oocyte quality and enhance embryonic development.

scholarly journals A Conserved Mitochondrial Chaperone-Protease Complex Involved in Protein Homeostasis

2021 ◽  
Vol 8 ◽  
Author(s):  
Mauro Serricchio ◽  
Peter Bütikofer
Keyword(s):  
Quality Control ◽  
Protein Complex ◽  
Elevated Temperatures ◽  
Protein Complexes ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Protein ◽  
Culture Conditions ◽  
Inner Mitochondrial Membrane ◽  
Knock Out ◽  
Mitochondrial Functions

Mitochondria are essential organelles involved in cellular energy production. The inner mitochondrial membrane protein stomatin-like protein 2 (SLP-2) is a member of the SPFH (stomatin, prohibitin, flotilin, and HflK/C) superfamily and binds to the mitochondrial glycerophospholipid cardiolipin, forming cardiolipin-enriched membrane domains to promote the assembly and/or stabilization of protein complexes involved in oxidative phosphorylation. In addition, human SLP-2 anchors a mitochondrial processing complex required for proteolytic regulation of proteins involved in mitochondrial dynamics and quality control. We now show that deletion of the gene encoding the Trypanosoma brucei homolog TbSlp2 has no effect on respiratory protein complex stability and mitochondrial functions under normal culture conditions and is dispensable for growth of T. brucei parasites. In addition, we demonstrate that TbSlp2 binds to the metalloprotease TbYme1 and together they form a large mitochondrial protein complex. The two proteins negatively regulate each other’s expression levels by accelerating protein turnover. Furthermore, we show that TbYme1 plays a role in heat-stress resistance, as TbYme1 knock-out parasites displayed mitochondrial fragmentation and loss of viability when cultured at elevated temperatures. Unbiased interaction studies uncovered putative TbYme1 substrates, some of which were differentially affected by the absence of TbYme1. Our results support emerging evidence for the presence of mitochondrial quality control pathways in this ancient eukaryote.

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