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 Characterization of mitochondrial replication and transcription control during rat early development in vivo and in vitro

Reproduction ◽  
2007 ◽  
Vol 133 (2) ◽  
pp. 423-432 ◽  
Author(s):  
Yuichi Kameyama ◽  
France Filion ◽  
Jae Gyu Yoo ◽  
Lawrence C Smith
Keyword(s):  
Early Development ◽  
Copy Number ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Mrna Levels ◽  
Mtdna Copy Number ◽  
Mitochondrial Transcription ◽  
Mtdna Replication

In vitroculture (IVC), used in assisted reproductive technologies, is a major environmental stress on the embryo. To evaluate the effect of IVC on mitochondrial transcription and the control of mtDNA replication, we measured the mtDNA copy number and relative amount of mRNA for mitochondrial-related genes in individual rat oocytes, zygotes and embryos using real-time PCR. The average mtDNA copy number was 147 600 (±3000) in metaphase II oocytes. The mtDNA copy number was stable throughoutin vivoearly development and IVC induced an increase in mtDNA copy number from the 8-cell stage onwards.GapdmRNA levels vary during early development and IVC did not change the patterns of these housekeeping gene transcripts.PolrmtmRNA levels did not vary during early development up to the morula stage but increased at the blastocyst stage. IVC induced the up-regulation ofPolrmtmRNA, one of the key genes regulating mtDNA transcription and replication, at the blastocyst stage. An increase inmt-Nd4mRNA preceded the blastocyst-related event observed in nuclear-encodedGapdandPolrmt, suggesting that the expression of mitochondrial encoded genes is controlled differently from nuclear encoded genes. We conclude that the IVC system can perturb mitochondrial transcription and the control of mtDNA replication in rat embryos. This perturbation of mtDNA regulation may be responsible for the abnormal physiology, metabolism and viability ofin vitro-derived embryos.

scholarly journals Selection of Ovine Oocytes by Brilliant Cresyl Blue Staining

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Liqin Wang ◽  
Jiapeng Lin ◽  
Juncheng Huang ◽  
Jing Wang ◽  
Yuncheng Zhao ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
G6pd Activity ◽  
Blue Staining ◽  
Animal Cloning ◽  
Cresyl Blue ◽  
Deep Blue ◽  
Glucose 6 Phosphate Dehydrogenase

Sheep oocytes derived from the ovaries collected from the slaughterhouse are often used for research onin vitroembryo production, animal cloning, transgenesis, embryonic stem cells, and other embryo biotechnology aspects. Improving thein vitroculture efficiency of oocytes can provide more materials for similar studies. Generally, determination of oocyte quality is mostly based on the layers of cumulus cells and cytoplasm or cytoplasm uniformity and colors. This requires considerable experience to better identify oocyte quality because of the intense subjectivity involved (Gordon (2003), Madison et al. (1992) and De Loos et al. (1992)). BCB staining is a function of glucose-6-phosphate dehydrogenase (G6PD) activity, an enzyme synthesized in developing oocytes, which decreases in activity with maturation. Therefore, unstained oocytes (BCB−) are high in G6PD activity, while the less mature oocytes stains are deep blue (BCB+) due to insuffcient G6PD activity to decolorize the BCB dye.

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.

Meiotic progression, mitochondrial features and fertilisation characteristics of porcine oocytes with different G6PDH activities

2010 ◽  
Vol 22 (5) ◽  
pp. 830 ◽  
Author(s):  
István Egerszegi ◽  
Hannelore Alm ◽  
József Rátky ◽  
Bassiouni Heleil ◽  
Klaus-Peter Brüssow ◽  
...  
Keyword(s):  
Developmental Competence ◽  
Mitochondrial Activity ◽  
G6pdh Activity ◽  
Cresyl Blue ◽  
Meiotic Progression ◽  
Germinal Vesicles ◽  
Nuclear Development ◽  
Chromatin Configuration ◽  
Glucose 6 Phosphate Dehydrogenase

The aim of the present study was to investigate the developmental competence, mitochondrial characteristics and chromatin status of immature follicular porcine oocytes selected for their glucose-6-phosphate dehydrogenase (G6PDH) activity by brilliant cresyl blue (BCB) staining. In Experiment 1, the oocyte parameters were determined in parallel right after BCB staining (T0), after 22 h of in vitro maturation (IVM) (T22) and after 44 h of IVM (T44) (n = 496). BCB-stained oocytes (BCB+) at T0 were characterised by fibrillated chromatin filaments in their germinal vesicles (GV) and diakinesis stages whereas unstained (BCB–) oocytes at T0 contained in their GV mainly condensed stages of chromatin (P < 0.05). After 22 h of IVM BCB+ oocytes showed a prominent chromatin configuration of metaphase I and after 44 h the majority developed a M II nuclear configuration in contrast to the BCB– group (P < 0.0001). Differences were also observed between the two oocyte populations in their mitochondrial activity (P < 0.05). At the beginning of IVM BCB+ oocytes were characterised by high mitochondrial activity in their cytoplasm. The BCB+ oocytes showed clear visible homogenous distributions of mitochondria (P < 0.005) and contained more aggregated clusters of mitochondria in contrast to BCB– oocytes (P < 0.005). In Experiment 2, 318 oocytes were tested for their G6PDH activity and introduced to IVM and IVF. Only oocytes from the BCB+ group, which were matured after 44 h up to the stage of M II (81.6%) were fertilised (17.4%), penetrated (46%) or activated (15.6%) after IVF. These results indicate a relationship between the G6PDH activity of porcine oocytes before IVM and their subsequent nuclear development, mitochondrial activity and aggregation.

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.

scholarly journals The Number of LH Receptor could Predict the Success of Oocyte Maturity in the Process of In Vitro Maturation

2016 ◽  
Author(s):  
Adek Amansyah
Keyword(s):  
In Vitro Maturation ◽  
Clinical Laboratory ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Maturity ◽  
Lh Receptor ◽  
First Polar Body ◽  
Significant Difference

Objective: To evaluate the relationship between the number of LH receptor and the success of oocyte maturity in the process of in vitro maturation (IVM). Method: This experimental study was conducted in the Permata Hati Infertility Clinical Laboratory, Dr. Sardjito General Hospital, Yogyakarta, with the samples of 300 oocytes obtained through collecting immature cow’s oocytes from the abattoir and grouped the oocytes into 3 (three) groups based on the pattern of oocyte cumulus cells on the vesicle germinal stage 2 - 8 mm with three layers of cumulus cell. The sample of the cumulus cells from these three groups were taken and the LH receptor examination was done with immunohistochemistry. After that, the IVM process was performed to the three groups and its development for 24 hours was evaluated. Its maturation quality was evaluated with the emergence of the first polar body (1PB) and compared to the other groups and related to the number of LH receptor in the three groups. Result: The result of this study indicated that the oocyte cumulus cells showed a difference of function during IVM process. The maturity rate in this study showed that the number of LH receptor was related to the morphological pattern of oocyte cumulus cells with oocyte maturity. The maturity of the cumulus cells which 100% covered the oocyte was higher than that of the cumulus cells which > 50% and < 30% covered the oocytes, namely, 74% compared to 60% and 12%. The result of this study also showed that the average number of LH receptors in the three groups (A, B, and C) was 183.4, 78.8, and 24.0 respectively. A significant difference was found in the three groups (p < 0.0001). When related to IVM maturity, this difference showed that the bigger number of oocyte cumulus cells influenced the oocyte maturity. Conclusion: The number of LH receptor can be used as a prediction to determine the success of oocyte maturation in the process of in vitro maturation. [Indones J Obstet Gynecol 2013; 1-4:183-7] Keywords: IVM, LH receptor, oocyte cumulus cell

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.

Fertilization and early embryonic development of in vitro matured metaphase I oocytes in patients with unexpected low oocyte maturity rate

Zygote ◽  
2021 ◽  
pp. 1-5
Author(s):  
Nafiye Yılmaz ◽  
Şebnem Özyer ◽  
Derya Taş ◽  
Mehmet Caner Özer ◽  
Ayten Türkkanı ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Fertilization Rate ◽  
Early Embryonic Development ◽  
Oocyte Maturity ◽  
Significant Difference ◽  
Maturation Rate ◽  
Developmental Capacity ◽  
Metaphase I

Summary To determine the fertilization and embryonic potential of immature metaphase I (MI) oocytes in patients with low oocyte maturity rate in whom the percentage of mature oocytes obtained was less than 75% of the total retrieved ones. In vivo matured metaphase II (MII) oocytes (MII-ICSI, n = 244), and in vitro matured MI oocytes (MI-MII-ICSI, n = 202) underwent an intracytoplasmic sperm injection (ICSI) procedure. Maturation rate, fertilization rate and early embryonic development were compared in both groups. In total, 683 oocytes were collected from 117 ICSI cycles of 117 patients. Among them, 244 (35.7%) were mature MII and 259 (37.9%) were MI after the denudation process. Of those 259 MI oocytes, 202 (77.9%) progressed to MII oocytes after an incubation period of 18–24 h. The maturation rate was 77.9%. Fertilization rate was found to be significantly higher in the rescued in vitro matured MI oocyte group when compared with the in vivo matured MII oocyte group (41.6% vs 25.8%; P = 0.0006). However, no significant difference was observed in terms of cleavage rates on days 2 and 3 between the groups (P = 0.9126 and P = 0.5031, respectively). There may be unidentified in vivo factors on the oocyte maturation causing low developmental capacity in spite of high fertilization rates in the group of patients with low oocyte maturity rate. Furthermore, studies are needed to determine the appropriate culture characteristics as well as culture period and ICSI timing of these oocytes.

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.

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.

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