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 A Case-control Study on the Association of Mitochondrial Transcription Factor a Gene +35G/C Polymorphism and Mitochondrial DNA Copy Number with the Risk of Endometriosis in Indian Women

2021 ◽  
pp. 60-67
Author(s):  
Himabindu Beeram ◽  
Tumu Venkat Reddy ◽  
Suresh Govatati ◽  
Swapna Siddamalla ◽  
Mamata Deenadayal ◽  
...  
Keyword(s):  
Copy Number ◽  
Case Control Study ◽  
Case Control ◽  
Mtdna Copy Number ◽  
Chain Reaction ◽  
Indian Women ◽  
Mitochondrial Transcription Factor ◽  
Mitochondrial Transcription Factor A ◽  
Polymerase Chain ◽  
Control Study

Aim: The Mitochondrial transcription factor A (TFAM) and mitochondrial (mt) DNA copy number variations are known to contribute in disease development. Genetic factors play an important role in the development of endometriosis. Therefore, this case–control study aimed to analyze the association of TFAM+35G/C polymorphism and mitochondrial copy number with the risk of endometriosis in Indian women. Study Design: This study was carried out on 418 subjects including 200 endometriosis cases and 218 controls. Methodology: Genotyping of TFAM +35G/C polymorphism (rs1937) was carried out by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Quantification of mtDNA copy number was carried out using a real time quantitative polymerase chain reaction (qRT-PCR). Place and Duration of Study: Department of Biochemistry, Osmania University, 2014 to 2020. Results: TFAM genotype as well as allele distributions were all in Hardy-Weinberg equilibrium. The results indicated a significant reduction of GG genotype frequency (P=0.009), high ‘C’ allele frequency (P=0.017) and significantly decreased mtDNA copy number in endometriosis cases compared to controls (P= 0.0001). Conclusion: Present study revealed a statistically significant association of decreased GG genotype of TFAM +35G/C polymorphism and mtDNA copy number with the risk of developing endometriosis in Indian women.

264 MITOCHONDRIAL DNA COPY NUMBER IN OOCYTES OF PREPUBERTAL AND CYCLIC GILTS

2011 ◽  
Vol 23 (1) ◽  
pp. 230
Author(s):  
P. Pawlak ◽  
E. Pers-Kamczyc ◽  
D. Lechniak-Cieslak
Keyword(s):  
Mitochondrial Dna ◽  
Copy Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Published Data ◽  
Mtdna Copy Number ◽  
Final Size ◽  
Domestic Species ◽  
Mitochondrial Dna Copy Number ◽  
Wide Range

In many domestic species (pig, cow, sheep), oocytes from prepubertal females show impaired quality when compared with those from adult animals. Incomplete cytoplasmic maturation is thought to be the main factor responsible for reduced developmental competence of embryos derived from prepubertal oocytes. The status of ooplasm maturation is also reflected by the copy number of mitochondrial DNA (mtDNA). Because replication of mtDNA ceases when oocytes reach their final size and occurs again at the blastocyst stage, the mtDNA copy number is a proved marker of oocyte quality in the pig (El Shourbagy et al. 2006 Reproduction 131, 233–245). The number of mtDNA copies in the grown oocyte is crucial to sustain the first embryonic divisions. To increase the rate of good-quality blastocysts, oocytes of domestic animals have been evaluated by the brilliant cresyl blue test (BCB). According to El Shourbagy et al. (2006), more competent BCB+ oocytes possess higher copy number of mtDNA (on average 222 446) than do their BCB– counterparts (115 352). However, there are no published data on the variation in mtDNA copy number in oocytes derived from ovaries of prepubertal (NCL) and cyclic (CL) gilts. Ovaries of NCL and CL gilts were collected in a local slaughterhouse. Cumulus–oocyte complexes (COC) were aspirated from nonatretic follicles 2 to 6 mm in diameter and evaluated morphologically. Only COC with a proper morphology were subjected to the BCB test. A group of non-BCB-treated COC served as control. Four groups of COC were collected: BCB+ (CL, NCL) and control (CL, NCL). Follicular cells attached to oocytes were removed by pipetting, and completely denuded gametes were individually frozen in liquid nitrogen. Analysis of the mtDNA copy number included isolation of the total DNA followed by amplification of the Cytochrome b (CYTB) gene by real-time PCR (one copy per one mitochondrial genome). Differences in mtDNA copy number among experimental groups were evaluated by Student’s t-test. To date, 30 BCB+ oocytes have been analysed individually (15 CL and 15 NCL). The analysed parameter varied in a wide range from 79 852 to 522 712 copies in CL oocytes and from 52 270 to 287 852 copies in NCL oocytes. Oocytes from cyclic gilts contained significantly more mtDNA copies (on average 267 524) than did gametes of prepubertal females (179 339; P < 0.05). The data on the mtDNA copy number in the control oocytes are currently under investigation. The preliminary results indicate that impaired oocytes quality of prepubertal gilts may be also attributed to the reduced copy number of mtDNA. This project was sponsored by MSHE Poland (grant no. 451/N-COST/2009/0).

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&lt;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 Mitochondria directly influence fertilisation outcome in the pig

Reproduction ◽  
2006 ◽  
Vol 131 (2) ◽  
pp. 233-245 ◽  
Author(s):  
Shahinaz H El Shourbagy ◽  
Emma C Spikings ◽  
Mariana Freitas ◽  
Justin C St John
Keyword(s):  
Copy Number ◽  
Mtdna Copy Number ◽  
Oocyte Maturity ◽  
Oocyte Competence ◽  
Cresyl Blue ◽  
Significant Difference ◽  
Mtdna Replication ◽  
Mitochondrial Number ◽  
Glucose 6 Phosphate Dehydrogenase

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.

scholarly journals HEK293T Cells with TFAM Disruption by CRISPR-Cas9 as a Model for Mitochondrial Regulation

Life ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 22
Author(s):  
Vanessa Cristina de Oliveira ◽  
Kelly Cristine Santos Roballo ◽  
Clésio Gomes Mariano Junior ◽  
Sarah Ingrid Pinto Santos ◽  
Fabiana Fernandes Bressan ◽  
...  
Keyword(s):  
Copy Number ◽  
Control Cell ◽  
Mtdna Copy Number ◽  
Mitochondrial Transcription Factor ◽  
Cell Clones ◽  
Key Factor ◽  
Mitochondrial Transcription Factor A ◽  
Vitro Model ◽  
Mitotracker Red

The mitochondrial transcription factor A (TFAM) is considered a key factor in mitochondrial DNA (mtDNA) copy number. Given that the regulation of active copies of mtDNA is still not fully understood, we investigated the effects of CRISPR-Cas9 gene editing of TFAM in human embryonic kidney (HEK) 293T cells on mtDNA copy number. The aim of this study was to generate a new in vitro model by CRISPR-Cas9 system by editing the TFAM locus in HEK293T cells. Among the resulting single-cell clones, seven had high mutation rates (67–96%) and showed a decrease in mtDNA copy number compared to control. Cell staining with Mitotracker Red showed a reduction in fluorescence in the edited cells compared to the non-edited cells. Our findings suggest that the mtDNA copy number is directly related to TFAM control and its disruption results in interference with mitochondrial stability and maintenance.

217 TRANSCRIPT ABUNDANCE OF CATHEPSIN GENES IN CUMULUS CELLS AS A MARKER OF CATTLE OOCYTE QUALITY

2013 ◽  
Vol 25 (1) ◽  
pp. 257
Author(s):  
E. Warzych ◽  
A. Wolc ◽  
A. Cieslak ◽  
D. Lechniak-Cieslak
Keyword(s):  
Real Time Pcr ◽  
Copy Number ◽  
Transcript Abundance ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Mtdna Copy Number ◽  
Cdna Synthesis ◽  
Fa Composition ◽  
Grade 3

Dynamics of follicular growth and atresia is closely connected with apoptosis. Cathepsins (CTS) are involved in diverse biological functions, whereas one member of this family, cathepsin B, plays a major regulatory role in the process of apoptosis. Oocyte quality is a complex trait shaped by the follicular components (e.g. cumulus cells, CC; follicular fluid, FF). A negative relationship between relative transcript abundance (RA) of CTSB, CTSS, and CTSZ genes in CC with the quality of corresponding oocytes was reported in cattle (Bettegowda et al. 2008). Fatty acid (FA) composition of the FF and mtDNA copy number in the oocyte are other markers of oocyte quality. Therefore, in this study, we analysed relations between selected traits of the 3 follicular components (FF, CC, oocyte) within the individual follicle with the focus on oocyte quality. The experiment was based on cumulus–oocyte-complexes (COC) and FF obtained from individual follicles of slaughterhouse ovaries. Each follicle was measured and assigned into 1 of 3 classes (small <6 mm; medium 6 to 8 mm; large >8 mm). The COC morphology (grades 1 to 4) was evaluated according to Stojkovic et al. (2001). The following analyses were performed: CC, mRNA abundance of CTSB, CTSS, CTSZ, and CTSK genes (real-time PCR, 100 replicates, ACTB as a reference gene); the oocyte, mtDNA copy number (real-time PCR, 93 replicates, COX1 gene); and FF and FA composition (gas chromatography). The following procedures were employed: total RNA isolation, mirVana Paris Kit (Ambion); total DNA isolation, High Pure PCR Template Preparation Kit (Roche, Indianapolis, IN, USA); cDNA synthesis, Transcriptor High Fidelity cDNA Synthesis Kit (Roche); and the standard curve method, to analyse the qPCR data. For statistical analysis, the Kruskal-Wallis test as well as Spearman rank correlation were applied. The highest RA of CTSB gene was noted in CC from the grade 3 COC (P < 0.05), whereas that of CTSK and CTSZ genes in CC from the grade 4 COC (P < 0.01). Because grade 3 and 4 COC are not suitable for IVM, we assumed that high RA of CTS gene in CC may indicate reduced quality of the corresponding oocyte. Surprisingly, the highest RA for CTSB gene was observed in CC from the medium follicles (P < 0.05). Significant (P < 0.05) correlations were estimated between the following: RA of CTSB gene in CC and mtDNA copy number in the oocyte (r = 0.27), RA of CTSB gene in CC and C18.3 n-3 concentration in FF (r = 0.32), RA of CTSZ gene in CC and C18.3 n-3 concentration in FF (r = 0.37), as well as RA of CTSZ gene in CC and n-3 concentration in FF (r = 0.34). Although an increase in RA of the CTS genes in CC was accompanied by the inferior oocyte morphology, it was also correlated with higher mtDNA copy number in the oocyte and FA content in FF. The last 2 features have been previously attributed to oocytes of better quality, what contrasts with the high RA of the CTS genes. Thus, higher RA of CTS genes within CC may not mark the bovine oocyte of reduced quality. Funding–National Science Center, grant no. N N302 604438.

102 REPEATED OVARIAN STIMULATIONS BY EXOGENOUS GONADOTROPIN COULD ALTER ATP CONTENT AND MITOCHONDRIAL DISTRIBUTION IN MOUSE OOCYTES

2013 ◽  
Vol 25 (1) ◽  
pp. 198
Author(s):  
Y. Kameyama ◽  
G. Shimoi ◽  
S. Kubo ◽  
R. Hashizume
Keyword(s):  
Treatment Group ◽  
Mitochondrial Function ◽  
Copy Number ◽  
Developmental Competence ◽  
Atp Content ◽  
Mtdna Copy Number ◽  
The Third ◽  
Significant Difference ◽  
Mitochondrial Distribution ◽  
The Mean

Ovarian stimulation (OS) by exogenous gonadotropin enhances the availability of mammalian oocytes but compromises their developmental competence (Combelles and Albertini 2003 Biol. Reprod. 68, 812–821). Recently, several studies have reported mitochondrial function-related abnormalities in oocytes after single and repeated OSs. Because mitochondria can directly influence fertilization outcomes (El Shourbagy et al. 2006 Reproduction 131, 233–245), this study aimed to determine the relationship between mitochondria-related parameters and developmental competence on the basis of ATP content, mitochondrial DNA (mtDNA) copy number, mitochondrial distribution, and IVF results for oocytes after repeated OSs. Ovulated oocytes were recovered from ICR female mice treated with 7.5 IU of eCG and 5 IU of hCG at an interval of 48 h in 1 (control) to 3 stimulation cycles, which were performed at intervals of either 5 or 10 d (n = 15–25 in each treatment group). The ATP content in oocytes was determined using a luminometer and commercial kits (BacTiter-Glo; Promega, WI, USA; n = 15–29 in each treatment group). The mtDNA copy number in oocytes was quantified by performing absolute quantification with real-time PCR (n = 4–8 in each treatment group). Mitochondrial distribution in oocytes stained by rhodamine123 was observed under a confocal microscope (n = 12–26 in each treatment group). These analyses were performed only for morphologically normal oocytes. The data were analyzed by one-way ANOVA, followed by Fisher’s least significant difference, or by the chi-square test. Some mice did not ovulate in the third stimulation cycle for both intervals (5-d interval, 32%; 10-d interval, 80%). The mean numbers of ovulated oocytes gradually decreased with progression of the stimulation cycles. The ATP content of the oocytes significantly decreased both in the second and third stimulation cycles, performed with a 5-d interval (control, 1.038 ± 0.117 pmol; second cycle, 0.852 ± 0.189 pmol; third cycle, 0.932 ± 0.272 pmol). The mean mtDNA copy number in oocytes did not change significantly but varied widely in the third stimulation cycle (control, 146 000 ± 21 000; 5-d interval, 135 000 ± 35 000; 10-d interval: 148 000 ± 50 000; mean ± SD). The mitochondrial staining patterns were classified into homogeneous, aggregation, and perinuclear accumulation. The rates of aggregation and perinuclear accumulation increased after repeated stimulation cycles. The blastocyst rates did not significantly differ among the treatment groups after IVF (75–82%). Repeated OSs not only decreased the number of ovulated oocytes but also caused changes related with mitochondrial function, even in the morphologically normal ovulated oocytes. Translocation of active mitochondria, which are associated with energy production, has some functional correlation with successful pre-implantation development (Suzuki et al. 2006 J. Mamm. Ova. Res. 23, 128–134). Changes in mitochondrial distribution might compensate for the negative effect of the decrease in ATP content and low mtDNA copy number after repeated OSs to help reach the blastocyst stage.

scholarly journals Tfam knockdown results in reduction of mtDNA copy number, OXPHOS deficiency and abnormalities in zebrafish embryos

2019 ◽  
Author(s):  
Auke BC Otten ◽  
Rick Kamps ◽  
Patrick Lindsey ◽  
Mike Gerards ◽  
Hélène Pendeville-Samain ◽  
...  
Keyword(s):  
Copy Number ◽  
Mrna Translation ◽  
Abnormal Development ◽  
Mtdna Copy Number ◽  
Zebrafish Model ◽  
Developmental Defects ◽  
Atp Production ◽  
Mitochondrial Transcription Factor ◽  
Copy Numbers ◽  
Mtdna Replication

ABSTRACTHigh mitochondrial DNA (mtDNA) copy numbers are essential for oogenesis and embryogenesis and correlate with fertility of oocytes and viability of embryos. To understand the pathology and mechanisms associated with low mtDNA copy numbers, we knocked down mitochondrial transcription factor A (Tfam), a regulator of mtDNA replication, during early zebrafish development. Reduction of Tfam using a splice-modifying morpholino (MO) resulted in a 42%±4% decrease in mtDNA copy number in embryos at 4 days post fertilization. Morphant embryos displayed abnormal development of the eye, brain, heart and muscle, as well as a 50%±11% decrease in ATP production. Transcriptome analysis revealed a decrease in protein-encoding transcripts from the heavy strand of the mtDNA. In addition, various RNA translation pathways were increased, indicating an upregulation of nuclear and mitochondria-related translation. The developmental defects observed were supported by a decreased expression of pathways related to eye development and haematopoiesis. The increase in mRNA translation might serve as a compensation mechanism, but appears insufficient during prolonged periods of mtDNA depletion, highlighting the importance of high mtDNA copy numbers for early development in zebrafish.SUMMARY STATEMENTThe first tuneable zebrafish model used to characterize the effect of a reduced mtDNA copy number and resulting OXPHOS deficiency on zebrafish embryonic development.

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