Regulated mitochondrial DNA replication during oocyte maturation is essential for successful porcine embryonic development.

Cellular ATP is mainly generated through mitochondrial oxidative phosphorylation, which is dependent on mitochondrial DNA (mtDNA). We have previously demonstrated the importance of oocyte mtDNA for porcine and human fertilization. However, the role of nuclear-encoded mitochondrial replication factors during oocyte and embryo development is not yet understood. We have analyzed two key factors, mitochondrial transcription factor A (TFAM) and polymerase gamma (POLG), to determine their role in oocyte and early embryo development. Competent and incompetent oocytes, as determined by brilliant cresyl blue (BCB) dye, were assessed intermittently during the maturation process for TFAM and POLG mRNA using real-time RT-PCR, for TFAM and POLG protein using immunocytochemistry, and for mtDNA copy number using real-time PCR. Analysis was also carried out following treatment of maturing oocytes with the mtDNA replication inhibitor, 2',3'-dideoxycytidine (ddC). Following in vitro fertilization, preimplantation embryos were also analyzed. Despite increased levels of TFAM and POLG mRNA and protein at the four-cell stage, no increase in mtDNA copy number was observed in early preimplantation development. To compensate for this, mtDNA appeared to be replicated during oocyte maturation. However, significant differences in nuclear-encoded regulatory protein expression were observed between BCB(+) and BCB(-) oocytes and between untreated oocytes and those treated with ddC. These changes resulted in delayed mtDNA replication, which correlated to reduced fertilization and embryonic development. We therefore conclude that adherence to the regulation of the timing of mtDNA replication during oocyte maturation is essential for successful embryonic development

Pre-hybridisation: an efficient way of suppressing endogenous biotin-binding activity inherent to biotin–streptavidin detection system

Endogenous biotin or biotinylated protein binding activity is a major drawback to biotin-avidin/streptavidin detection system. The avidin/streptavidin conjugate used to detect the complex of the biotinylated secondary antibody and the primary antibody binds to endogenous biotin or biotinylated proteins leading to non-specific signals. In Western blot, the endogenous biotin or biotinylated protein binding activity is usually manifested in the form of ~72kDa, ~75kDa and ~150kDa protein bands, which often mask the signals of interest. To overcome this problem, a method based on prior hybridisation of the biotinylated secondary antibody and the streptavidin conjugate was developed. The method was tested alongside the conventional biotin-streptavidin method on proteins extracted from zebrafish (Danio rerio) embryos. Results showed that the newly developed method efficiently suppresses the endogenous biotin or biotinylated protein binding activity inherent to the biotin-streptavidin detection system

Short-term chilled storage of zebrafish (Danio rerio) embryos in cryoprotectant as an alternative to cryopreservation.

As zebrafish embryos have never been cryopreserved, we developed a protocol to store zebrafish embryos (50% epiboly-5.3 hour post fertilization) for up to 18 h at 0°C. Initial experiments to optimize the cryoprotectant (CPA) solution demonstrated improved embryo hatching rate following chilling at 0°C for 18 h with 1 M MeOH+0.1 M sucrose (56 ± 5%) compared with other combinations of methanol (0.2-0.5 M) and sucrose (0.05-0.1 M). This combination of CPAs that protects against chilling injury was further tested to assess its impact on sox gene and protein expression. Significant decreases in sox3 gene expression were observed in hatched embryos that had been chilled for 18 h in 1 M MeOH+0.1 sucrose compared with non-chilled controls, however the expression of both sox2 and sox3 proteins was unaffected. Significant decreases in sox2 protein expression were, however, observed in embryos that had been chilled without CPAs and these embryos also had lower hatching rates than those chilled with the optimal CPA solution. We, therefore, conclude that the CPA combination of 1 M MeOH+0.1 M sucrose facilitates chilled storage of early stage (50% epiboly) zebrafish embryos for up to 18 h without compromising transcriptional response

Effects of the slow cooling during cryopreservation on the survival and morphology of Taiwan shoveljaw carp (Varicorhinus barbatulus) spermatozoa

Over the past decades, pollution, overfishing, and habitat degradation have driven the population size of Taiwan shoveljaw carp down markedly in Taiwan. Cryopreservation is a useful tool which could be used to maintain genetic resources to protect and preserve this endemic species. Four cryoprotectants [dimethyl sulphoxide (DMSO), dimethylacetamide (DMA), glycerol and methanol] and six freezing rates (0.5, 1, 2, 4, 8, 16 °C min-1) were tested in order to develop an optimal controlled slow-freezing protocol for Taiwan shoveljaw carp spermatozoa. Samples were subsequently examined under the scanning electron microscope to reveal whether cryopreservation had affected their ultrastructural morphology. The highest survival rate (50.1 ± 2.0%) was observed with a freezing rate of 8 °C min-1 in 1M DMSO, using SYBR-14 + PI staining. Fertility and hatching rate results using frozen-thawed spermatozoa (90.2 ± 2.2% and 22.3 ± 2.5%, respectively) were not significantly different from results with fresh spermatozoa. After cryopreservation, 21.0 ± 1.6% of frozen-thawed spermatozoa had mid-piece swelling and rupture of the head. Cryopreservation might, therefore, slightly affect Taiwan shoveljaw carp spermatozoa in terms of morphological change. However, these alterations could be compensated by using large enough numbers of normally functioning frozen-thawed spermatozoa to achieve a standard equal to fresh spermatozoa. This is the first report of successful cryopreservation of Taiwan shoveljaw carp spermatozoa using a controlled slow-cooling method

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

Use of methanol as cryoprotectant and its effect on sox genes and proteins in chilled zebrafish embryos

Methanol is a widely used cryoprotectant (CPA) in cryopreservation of fish embryos, however little is known about its effect at the molecular level. This study investigated the effect of methanol on sox gene and protein expression in zebrafish embryos (50% epiboly) when they were chilled for 3. h and subsequently warmed and cultured to the hatching stages. Initial experiments were carried out to evaluate the chilling tolerance of 50% epiboly embryos which showed no significant differences in hatching rates for up to 6. h chilling in methanol (0.2-, 0.5- and 1. M). Subsequent experiments in embryos that had been chilled for 3. h in 1. M methanol and warmed and cultured up to the hatching stages found that sox2 and sox3 gene expression were increased significantly in hatched embryos that had been chilled compared to non-chilled controls. Sox19a gene expression also remained above control levels in the chilled embryos at all developmental stages tested. Whilst stable sox2 protein expression was observed between non-chilled controls and embryos chilled for 3. h with or without MeOH, a surge in sox19a protein expression was observed in embryos chilled for 3. h in the presence of 1. M MeOH compared to non-chilled controls and then returned to control levels by the hatching stage. The protective effect of MeOH was increased with increasing concentrations. Effect of methanol at molecular level during chilling was reported here first time which could add new parameter in selection of cryoprotectant while designing cryopreservation protocol

Mitochondrial DNA replication in pre-implantation embryonic development

All eukaryotic cells possess mitochondrial DNA (mtDNA), which is maternally inherited through the oocyte, its replication being regulated by nuclear-encoded replication factors. It was hypothesised that mtDNA replication is highly regulated in oocytes, pre-implantation embryos and embryonic stem cells (ESCs) and that this may be disrupted following nuclear transfer (NT). MtDNA copy number decreased between 2-cell and 8-cell staged porcine embryos and increased between the morula and expanded blastocyst stages, coinciding with increased expression of mtDNA replication factors. Competent porcine oocytes replicated their mtDNA prior to and during in vitro maturation to produce and maintain the 100000 mtDNA copies required for fertilisation. Those oocytes in which mtDNA replication was delayed had reduced developmental ability. Expression of pluripotency-associated genes decreased as murine ESCs differentiated into embryoid bodies, although expression of mtDNA replication factors did not increase until the stage equivalent to organogenesis. Cross-species NT embryos in which the donor cell-derived mtDNA was replicated produced decreased developmental outcomes compared to those in which no mtDNA replication took place. Disruption of the strict regulation of mtDNA replication that occurs during early embryogenesis, as is likely following NT, may therefore contribute to the reduced developmental ability of embryos produced using such techniques

Effect of chilling on sox2, sox3 and sox19a gene expression in zebrafish (Danio rerio) embryos

Zebrafish embryos have not been cryopreserved due to their structural limitations. Although embryo survival rates have been used as the measured outcome for most of the cryopreservation protocols studied, there are very limited data available at the molecular level. This study focused on the effect of chilling and subsequent warming on gene expression of sox2, sox3 and sox19a which play vital roles in the development of zebrafish embryos. A quantitative RT-PCR approach was used to investigate gene expression following chilling at 0°C for up to 180 min. The effect on gene expression was also studied during a 180 min warming period after chilling for 30 or 60 min. There were significant decreases in sox2 (up to 4-fold) and sox3 (up to 3-fold) expressions following chilling. Significant increases in gene expressions of sox2 (up to 2-fold), sox3 (up to 33-fold) and sox19a (up to 25-fold) were observed during warming in the embryos that had been chilled for 30 min. Similarly, significant increases were observed in sox2 (up to 3-fold) and sox3 (up to 2-fold) during warming in embryos that had been chilled for 60 min. These increases may be explained by compensation for the suppression observed during chilling and/or to activate repair mechanisms or maintain homeostasis