Accumulation of mitochondrial DNA during oogenesis in Xenopus laevis

The mitochondrial DNA (mtDNA) content of Xenopus laevis oocytes at various stages of oogenesis has been determined by molecular hybridization with ³H-labeled complementary RNA (cRNA). The previtellogenic oocyte less than 250 μm in diameter (stage 1) contains 0.95 ± 0.47 ng of mtDNA. Accumulation of mtDNA proceeds until stage 4 (500–750 μm diameter oocyte), by which time a steady-state level of 4.28 ± 0.40 ng/oocyte is attained. Using the hybridization assay, the stage 6 (full-grown) Xenopus oocyte contains 4.51 ± 0.69 ng of mtDNA, compared to the previously reported value of 3.8 ng determined by direct measurement on the unfertilized egg. There appears to be a reasonable correlation, therefore, between the termination of mtDNA accumulation and the dispersal of the juxtanuclear, mitochondrial aggregate (Balbiani body) at the onset of vitellogenesis in Xenopus. It is concluded that the enormous complement of oocyte mitochondria is accumulated well before the end of oocyte growth and is maintained at a constant level during the remainder of oogenesis, through maturation, fertilization, and on into early development.     

Calmodulin synthesis and accumulation during oogenesis and maturation of Xenopus laevis oocytes

The calmodulin levels in stage 6 Xenopus oocytes averaged 89 +/- 24 (SD) ng/oocyte and had largely accumulated by stage 3 of oogenesis. From stage 3 to early stage 6, calmodulin levels did not increase further. However, in large stage 6 oocytes (greater than 1.25 mm diam) calmodulin levels again rose to a level as high as 121 ng/oocyte. Calmodulin levels did not change during the maturation of stage 6 oocytes and the results of measurements on animal and vegetal oocyte halves from control and mature oocytes showed no evidence of a redistribution of calmodulin during maturation. Measurements of calmodulin synthesis in stages 1 and 2 oocytes, stage 4 oocytes, and stage 6 oocytes indicated that calmodulin was being synthesized continuously during oogenesis and that the rate of synthesis increased during oogenesis. In stage 1 and 2 oocytes (combined), the synthesis rate was 3.5 pg/hr/oocyte; in stage 4 oocytes it was 48 pg/hr/oocyte, and in large stage 6 oocytes the rate had increased to 160 pg/hr/oocyte. These changes in the rates of synthesis were discussed as they relate to the pattern of calmodulin accumulation during oogenesis.     

Cytophotometric and autoradiographic evidence for functional apomixis in a gynogenetic fish,Poecilia formosa and its related,triploid unisexuals

Summary Amounts of DNA in individual Feulgen-stained nuclei from squash preparations of ovaries and testes from wild-caught and laboratory-reared stocks of Poecilia spp. were determined with an integrating microdensitometer. The DNA content of primary spermatocytes (4C) at zygotene, pachytene, or at metaphase I (3.3–3.4 pg) was approximately twice that found in secondary spermatocytes (2C) and four times that found for young spermatids (1C). Rarely, mature sperm were found with 2C DNA amounts. Nuclei from follicular epithelium and oogonia from both bisexual and diploid unisexual fish contained about 1.6–1.7 pg DNA; whereas, the DNA content of primary oocyte nuclei was about 3.5–3.7 pg DNA, indicating that just one cycle of chromosomal replication had occurred in these cells during the period of DNA synthesis before the visible onset of meiotic prophase. Similar results were obtained for triploid unisexuals whose 6C primary oocyte nuclei contained 5.0–5.1 pg DNA, which was twice the DNA content of 3C oogonia and follicular epithelial cells (2.4–2.5 pg DNA). Autoradiographic studies, designed to monitor the incorporation of ³H-thymidine by oogonia and primary oocytes in vivo and in vitro, also showed that there is no additional synthesis of DNA during the course of meiotic prophase in these unisexual fish. Therefore, we conclude that apomixis, not endoreduplication, is the cytological basis of reproduction in Poecilia formosa and its related, triploid biotypes.     

Regulation of oocyte mitochondrial DNA copy number by follicular fluid, EGF, and neuregulin 1 during in vitro maturation affects embryo development in pigs

Little is known about mitochondrial DNA (mtDNA) replication during oocyte maturation and its regulation by extracellular factors. The present study determined the effects of supplementation of maturation medium with porcine follicular fluid (pFF; 0, 10%, 20%, and 30%) on mtDNA copy number and oocyte maturation in experiment 1; the effects on epidermal growth factor (EGF; 10 ng/mL), neuregulin 1 (NRG1; 20 ng/mL), and NRG1 + insulin-like growth factor 1 (IGF1; 100 ng/mL + NRG1 20 ng/mL), on mtDNA copy number, oocyte maturation, and embryo development after parthenogenic activation in experiment 2; and effects on embryo development after in vitro fertilization in experiment 3. Overall, mtDNA copy number increased from germinal vesicle (GV) to metaphase II (MII) stage oocytes after in vitro maturation (GV: 167 634.6 ± 20 740.4 vs. MII: 275 131.9 ± 9 758.4 in experiment 1; P < 0.05; GV: 185 004.7 ± 20 089.3 vs. MII: 239 392.8 ± 10 345.3 in experiment 2; P < 0.05; Least Squares Means ± SEM). Supplementation of IVM medium with pFF inhibited mtDNA replication (266 789.9 ± 11 790.4 vs. 318 510.1 ± 20 377.4; P < 0.05) and oocyte meiotic maturation (67.3 ± 0.7% vs. 73.2 ± 1.2%, for the pFF supplemented and zero pFF control, respectively; P < 0.01). Compared with the control, addition of growth factors enhanced oocyte maturation. Furthermore, supplementation of NRG1 stimulated mitochondrial replication, increased mtDNA copies in MII oocytes than in GV oocytes, and increased percentage of blastocysts in both parthenogenetic and in vitro fertilized embryos. In this study, mitochondrial biogenesis in oocytes was stimulated during in vitro maturation. Oocyte mtDNA copy number was associated with developmental competence. Supplementation of maturation medium with NRG1 increased mtDNA copy number, and thus provides a means to improve oocyte quality and developmental competence in pigs.     

Cryopreservation of zebrafish (Danio rerio) oocytes using improved controlled slow cooling protocols

Cryopreservation of gametes provides a promising method to preserve fish genetic material. Previously we reported some preliminary results on cryopreservation of zebrafish (Danio rerio) oocytes using controlled slow cooling and determined the optimum cryoprotective medium and cooling rate for stage III zebrafish oocytes. In the present study, the effects of two different cryopreservation media, cryoprotectant removal method, final sample freezing temperature before LN₂ plunge, warming rate, and the post-thaw incubation time on oocyte viability were investigated. Commonly used cryoprotectant methanol and glucose were used in this study. Stage III zebrafish oocytes were frozen in standard culture medium 50% L-15 or in a sodium-free KCl buffer medium. Oocyte viability was assessed using trypan blue staining and ATP assay. The viability of oocytes frozen in KCl buffer was significantly higher than oocytes frozen in L-15 medium. The results also showed that fast thawing and stepwise removal of cryoprotectant improved oocyte survival significantly, with highest viability of 88.0 ± 1.7% being obtained immediately after rapid thawing when assessed by trypan blue staining. However, after 2 h incubation at 22 °C the viability of freeze-thawed oocytes decreased to 29.5 ± 5.1%. Results also showed that the ATP level in oocytes decreased significantly immediately after thawing. All oocytes became translucent after freezing which complicated the use of GVBD test (in vitro maturation of oocytes followed by observation of germinal vesicle breakdown which results in oocytes becoming translucent). New oocyte viability assessment methods are urgently needed.     

In vitro embryo production efficiency in cattle and its association with oocyte adenosine triphosphate content, quantity of mitochondrial DNA, and mitochondrial DNA haplogroup

Mitochondria have a broad range of functions that affect reproduction, and structural as well as quantitative variation in mtDNA has been associated with gamete quality and reproductive success. To investigate the mitochondria effect on in vitro embryo production, we collected oocytes by ultrasound-guided follicular aspiration from donor cows known to differ in the developmental capacity, measured by the blastocyst formation rate, of their oocytes. To evaluate the potential effects of mtDNA and mitochondrial function on oocyte quality, the donor cows' mtDNA control region was sequenced and, after pairwise comparisons of polymorphisms, animals were grouped into two major haplogroups. The number of mtDNA molecules per oocyte was quantified by real-time PCR, and the adenosine triphosphate (ATP) content was measured in each oocyte to identify variations between haplogroups. Overall, ATP stocks in oocytes of the two haplogroups differed significantly (P < 0.05; means +/- SEM) both at the germinal vesicle and metaphase II stages (2.8 +/- 0.06 pmol vs. 2.6 +/- 0.07 pmol and 2.9 +/- 0.1 pmol vs. 2.3 +/- 0.06 pmol, respectively). The proportion of development to blastocyst was significantly different between haplogroups (22.3 +/- 2.1 % vs. 36.7 +/- 2.9 %). The number of mtDNA molecules per oocyte was highly variable (377 327 +/- 14 104, ranging from 2.0 x 10(3) to 1.2 x 10(6)) but not significantly different between the two haplogroups; significant differences were observed between animals without any apparent relationship to blastocyst production. These data suggest that mitochondria and mtDNA haplogroup affect the developmental capacity of bovine oocytes in vitro.     

Effect of chilling on porcine germinal vesicle stage oocytes at the subcellular level

The potential subcellular consequence of chilling on porcine germinal vesicle (GV) stage oocytes was examined. Prior to in vitro maturation (IVM), Cumulus-oocyte complexes (COCs) freshly collected from antral follicles (3–6 mm in diameter) were evenly divided into four groups and immediately incubated in PVA-TL-HEPES medium at the temperature of 39 °C (control group), 23 °C (room temperature), 15 °C and 10 °C for 10 min, respectively. Following 42 h of IVM at 39 °C, the survival rates were examined. There was no significant difference between the survival rate of 23 °C chilled group and control group (77.92 and 91.89%), but the survival rate of 15 and 10 °C chilled group were significantly decreased (46.34 and 4.81%, P < 0.01). A further experiment on15 °C group showed that most oocytes died from 2 to 4 h of IVM. In order to investigate the effects of chilling on oocytes at the subcellular level, the control and 15 °C chilled group COCs fixed at different time points of the IVM cultures (2, 2.5, 3, 3.5 and 4 h of IVM) were prepared for transmission electron microscope (TEM) observation. As the result, compared with the control group, there were two significant changes in the ultrastructural morphology of 15 °C treatment group: (1) dramatic reduction of heterogeneous lipid, (2) disorganized mitochondria–endoplasmic reticulum–lipid vesicles (M–E–L) combination. These results indicate that 15 °C is a critical chilling temperature for porcine GV stage oocyte and the alteration of cellular chemical composition and the destruction of M–E–L combination maybe responsible for chilling injury of porcine oocyte at this stage.     

Effect of C-type natriuretic peptide pretreatment on in vitro bovine oocyte maturation

C-type natriuretic peptide (CNP) has been considered as a physiological meiotic inhibitor that stimulates the cGMP production by cumulus cell natriuretic peptide receptor 2 (NPR2), which inhibits oocyte phosphodiesterase type 3 activity and increases cAMP. In this study, we explored the effect of CNP pretreatment on the in vitro maturation (IVM) of bovine oocytes by examining changes in cleavage rate, blastocyst formation, mitochondrial DNA (mtDNA) copy number, reactive oxygen species (ROS) level, glutathione (GSH) content, and redox state. Our results showed that 200 nM CNP could effectively maintain meiotic arrest of bovine oocytes in vitro within 6 h. The two-step IVM system in which oocytes were pretreated with 200 nM CNP for 6 h and then cultured IVM for 28 h yielded a significantly (P < 0.05) increased blastocyst rate and cell number after in vitro fertilization (IVF) while compared to the conventional one-step IVM method. In addition, in comparison with the conventional 24-h matured oocyte, oocytes pretreated with 200 nM CNP for 6 h followed by 28 h IVM resulted in significantly (P < 0.05) higher mtDNA copy number and ROS levels in oocytes, while GSH level significantly (P < 0.05) decreased. Remarkably, regardless of treatment, no changes were observed in FAD++, NAD(P)H autofluorescence intensity, and redox ratio (FAD++/NAD(P)H) within the oocytes, maintaining a healthy metabolic equilibrium of redox throughout the two-step IVM. In conclusion, these results indicate that CNP pretreatment could dramatically improve the quality of bovine oocytes during in vitro maturation.     

Gene amplification in oocytes with 8 germinal vesicles from the tailed frog Ascaphus truei Stejneger

In the last 3 oogonial mitoses in Ascaphus truei all daughter nuclei remain in the same cell. The oocyte is 8-nucleate at the start of meiotic prophase and remains so until late in oogenesis when 7 of the nuclei disappear. All 8 nuclei in a single oocyte resemble one another with respect to size and chromatin distribution at all stages of meiotic prophase. Much of the Feulgen-positive material in pachytene nuclei is concentrated into one region of the nucleus. — All of the 8 germinal vesicles of yolky oocytes have a full set of lampbrush diplotene bivalents. Germinal vesicles from oocytes of up to 0.8 mm diameter have less than 100 nucleoli, some of which are multiple nucleoli in the sense that they have more than one core region. Each of the 8 nuclei in oocytes from one animal had about the same volume of nucleolar material. — Two values have been obtained for the amount of DNA in a diploid nucleus from Ascaphus. A biochemical estimate utilizing erythrocyte nuclei and the diphenylamine reaction yielded a value of 7.1 pg per nucleus. Microphotometry of erythrocyte nuclei stained with Feulgen's reagent gave a value of 8.2 pg per nucleus. — Microphotometric measurements of Feulgen-stained nuclei at various stages of meiotic prophase up to diplotene indicate that each nucleus synthesizes up to 5 pg of extrachromosomal DNA during and immediately after pachytene. This DNA is considered to be nucleolar. Autoradiography of nuclei from oocytes which had been incubated for 6h in ³H thymidine showed silver grains over pachytene and early diplotene nuclei only. In pachytene nuclei the silver grains overlaid that part of the nucleus where Feulgen-positive material was most concentrated. Most of the chromosomal material was unlabelled. — The significance of the 8-nucleate condition in Ascaphus oocytes is discussed, and the amount of nucleolar DNA synthesized at pachytene and of nucleolar material present in germinal vesicles is compared with corresponding situations in other amphibians.     

Mitochondrial DNA copy number in bovine oocytes and somatic cells

Restriction endonuclease analysis and direct nucleotide sequencing of bovine mitochondrial DNA have revealed a high apparent rate of sequence divergence between maternally related individuals. One possible mechanism that would account for the high rate involves nonuniform amplification and/or segregation of mitochondrial DNA during development of the oocyte. We report here experiments which quantitate the amount of mitochondrial DNA in the bovine oocyte as compared to bovine somatic cells. Total DNA was isolated from purified oocytes, separated by agarose gel electrophoresis, and immobilized on nitrocellulose filters. Hybridization with the complete mitochondrial DNA genome or cloned mitochondrial DNA restriction fragments revealed a 100-fold increase in oocyte mitochondrial DNA as compared to somatic cells. Developing oocytes contained about 4.5 pg or 2.6 × 10⁵ copies per cell, whereas primary bovine tissue culture cells contained 0.045 pg or 2.6 × 10³ copies per cell. These experiments demonstrate directly the amplification of mitochondrial DNA in mammalian oocytes and are consistent with models which could generate mitochondrial DNA polymorphisms by unequal amplification of mitochondrial genomes within an animal.     

Determination of oocyte membrane permeability coefficients and their application to cryopreservation in a rabbit model

Having an effective means to cryopreserve human oocytes would offer more flexibility in healthcare services for infertility patients, and obviate cryopreservation of preimplantation embryos. It is essential to establish good animal models for human oocyte cryopreservation and the rabbit is a good candidate. Attempts to improve oocyte cryopreservation are often empirical, with results often being irreproducible. Cryopreservation protocols may be optimized by modeling the changes in oocyte volume and the associated damages incurred during the addition and dilution of cryoprotective agents (CPA). The objectives of the current study were to determine cryobiological properties of rabbit oocytes, including the isotonic volume, osmotically inactive cell fraction (V_b), hydraulic conductivity (L_p), permeability (P_s) to dimethylsulfoxide (Me₂SO), ethylene glycol (EG), and glycerol (GLY) and to examine the correlation between cell volume excursions and viability. This has led to the development of the accumulative osmotic damage (AOD) model associated with the processes of CPA addition/dilution. Mature rabbit oocytes were perfused with 15% (V/V) CPA medium (dissolved in 1× PBS). The osmotic responses of the oocytes were videotaped. A two-parameter model was fit to the experimental data to determine the values of L_p and P_s. Oocyte volumes reached upon equilibration with 285, 600, 900, and 1200 mOsm (milliosmolal) solutions of non-permeating compounds were plotted in a Boyle van’t Hoff plot. The average radius of rabbit oocytes in an isotonic solution was determined to be 55.7 ± 1.2 μm (n = 16). The rabbit oocyte exhibited an “ideal” osmotic response in the range from iso-osmolity to 1200 mOsm. The V_b was determined to be 20% of the isotonic value with r² = 0.97. The values of L_p were determined to be 0.79 ± 0.26, 0.82 ± 0.22, and 0.64 ± 0.16 μm min⁻¹ atm⁻¹ and the P_s values were determined to be 2.9 ± 1.3, 2.7 ± 1.3, and 0.27 ± 0.18 × 10⁻³ cm min⁻¹ for Me₂SO, EG and GLY, respectively. There were no significant differences (p > 0.05) between values for L_p and P_S in the presence of the Me₂SO and EG. However, these values were significantly different from the values in presence of GLY. We calculated the AOD values of those oocytes that experienced the process of CPA additions/dilutions and found that these values were highly correlated to the development rates of these oocytes after parthenogenetic activation (r = −0.98).     

γ-glutamyl transpeptidase of spermatozoa may decrease oocyte glutathione content at fertilization in pigs

The presence of γ-glutamyl transpeptidase (GGT) in boar spermatozoa and the potential role of the GGT at sperm penetration were examined using in vitro matured porcine oocytes. In the first experiment, GGT of boar spermatozoa was examined using a histochemical stain. GGT was detected in the midpiece and the acrosome regions of boar spermatozoa. In the second experiment, porcine oocytes matured in vitro were injected with approximately 40 pl of 10 mM HEPES solution alone or HEPES containing 0.5 U/ml GGT or 1 mM guanosine-5′-0-(3′-thiotriphosphate) (GTP-γ-S; G-protein activator). When GGT was injected into oocytes, the incidence of oocytes activated (23.7 ± 1.4%) was not different (P > 0.05) from HEPES-injected controls (24.9 ± 1.3%) at 6 h after injection. Injected GTP-γ-S, however, activated 76.0 ± 5.3% of oocytes at 6 h after injection, but extrusion of the second polar body was very low (2.8 ± 4.8%). Total content of glutathione (GSH) and glutathione disulfide (GSSG) did not differ (P > 0.05) between GTP-γ-S injected oocytes (4.2 ± 0.7 pmol/oocyte) and noninjected oocytes (4.0 ± 0.1 pmol/oocyte) at 6 h after injection. However, the total content of GSH and GSSG was lower (P < 0.01) in GGT-injected oocytes (2.1 ± 0.2 pmol/oocyte) than HEPES-injected oocytes (3.4 ± 0.2 pmol/oocyte) at 6 h after injection. In the third experiment, in vitro matured porcine oocytes were injected with about 40 pl of 10 mM HEPES solution alone or HEPES containing 0.5 U/ml GGT and then inseminated. At 12 h after insemination, the incidence of male pronuclear formation was significantly lower in oocytes injected with GGT as compared with injected control oocytes. These results demonstrated that (1) GGT was present on the surface of spermatozoa, (2) total oocyte content of GSH and GSSG was decreased by microinjection of GGT but not by that of GTP-γ-S, and (3) male pronuclear formation was inhibited in GGT-injected oocytes. These results suggest that sperm GGT may be a limiting factor for male pronuclear formation in polyspermic oocytes. © 1996 Wiley-Liss, Inc.     

Potential Role for MATER in Cytoplasmic Lattice Formation in Murine Oocytes

Background

Mater and Padi6 are maternal effect genes that are first expressed during oocyte growth and are required for embryonic development beyond the two-cell stage in the mouse. We have recently found that PADI6 localizes to, and is required for the formation of, abundant fibrillar Triton X-100 (Triton) insoluble structures termed the oocyte cytoplasmic lattices (CPLs). Given their similar expression profiles and mutant mouse phenotypes, we have been testing the hypothesis that MATER also plays a role in CPL formation and/or function.

Methodology/Findings

Herein, we show that PADI6 and MATER co-localize throughout the oocyte cytoplasm following Triton extraction, suggesting that MATER co-localizes with PADI6 at the CPLs. Additionally, the solubility of PADI6 was dramatically increased in Mater^tm/tm oocytes following Triton extraction, suggesting that MATER is involved in CPL nucleation. This prediction is supported by transmission electron microscopic analysis of Mater^+/+ and Mater^tm/tm germinal vesicle stage oocytes which illustrated that volume fraction of CPLs was reduced by 90% in Mater^tm/tm oocytes compared to Mater^+/+ oocytes.

Conclusions

Taken together, these results suggest that, similar to PADI6, MATER is also required for CPL formation. Given that PADI6 and MATER are essential for female fertility, these results not only strengthen the hypothesis that the lattices play a critical role in mediating events during the oocyte-to-embryo transition but also increase our understanding of the molecular nature of the CPLs.     

Donor-host mitochondrial compatibility improves efficiency of bovine somatic cell nuclear transfer

Background  

The interaction between the karyoplast and cytoplast plays an important role in the efficiency of somatic cell nuclear transfer (SCNT), but the underlying mechanism remains unclear. It is generally accepted that in nuclear transfer embryos, the reprogramming of gene expression is induced by epigenetic mechanisms and does not involve modifications of DNA sequences. In cattle, oocytes with various mitochondrial DNA (mtDNA) haplotypes usually have different ATP content and can further affect the efficiency of in vitro production of embryos. As mtDNA comes from the recipient oocyte during SCNT and is regulated by genes in the donor nucleus, it is a perfect model to investigate the interaction between donor nuclei and host oocytes in SCNT.     

Germ-line mitochondria exhibit suppressed respiratory activity to support their accurate transmission to the next generation

Mitochondria are accurately transmitted to the next generation through a female germ cell in most animals. Mitochondria produce most ATP, accompanied by the generation of reactive oxygen species (ROS). A specialized mechanism should be necessary for inherited mitochondria to escape from impairments of mtDNA by ROS. Inherited mitochondria are named germ-line mitochondria, in contrast with somatic ones. We hypothesized that germ-line mitochondria are distinct from somatic ones. The protein profiles of germ-line and somatic mitochondria were compared, using oocytes at two different stages in Xenopus laevis. Some subunits of ATP synthase were at a low level in germ-line mitochondria, which was confirmed immunologically. Ultrastructural histochemistry using 3,3′-diaminobenzidine (DAB) showed that cytochrome c oxidase (COX) activity of germ-line mitochondria was also at a low level. Mitochondria in one oocyte were segregated into germ-line mitochondria and somatic mitochondria, during growth from stage I to VI oocytes. Respiratory activity represented by ATP synthase expression and COX activity was shown to be low during most of the long gametogenetic period. We propose that germ-line mitochondria that exhibit suppressed respiration alleviate production of ROS and enable transmission of accurate mtDNA from generation to generation.     

Replication of mitochondrial DNA in the early development of Misgurnus fossilis

Mitochondria isolated from Misgurnus fossilis embryos at various developmental stages were incubated with ³H-dTTP in vitro and the incorporation into mtDNA was determined. It has been found that the rate of mtDNA labeling increases exponentially with a doubling time of 7 hr from 0.01 pmole of ³H-dTMP/mg protein/hr in mitochondria from unfertilized eggs to 0.4 pmoles of ³H-dTMP/mg protein/hr in mitochondria of 35 hr embryos. The pool of intramitochondrial dTTP decreases 2.5 times during the first 10 hr after fertilization, then remains practically constant up to 35 hr of development. The rate of exogenous ³H-dTTP incorporation into the acid soluble pool of isolated mitochondria at two stages is approximately proportional to the pool size. Thus identical specific activities of ³H-dTTP inside mitochondria would be obtained even with pools of different sizes. We conclude that the increase of ³H-dTMP incorporation into mtDNA in development reflects genuine activation of mtDNA synthesis. As early as 6 hr after fertilization the bulk of the label incorporated into mtDNA is found in the fraction associated with covalently closed molecules. This pattern of labeling characteristic for replicating mtDNA is maintained throughout early development. In contrast such preferential label incorporation into the closed circular fraction was not found with mitochondria of unfertilized eggs. Closed mtDNA from unfertilized eggs contains not more than 1% of molecules with D-loops. In 35 hr embryos the corresponding value is equal to about 4%. Activation of mtDNA replication in embryogenesis is probably due to the activation of mechanisms responsible for the generation of primers for replication. DNA polymerase activity solubilized from mitochondria remains unchanged in the course of embryogenesis.     

Interspecies somatic cell nuclear transfer is dependent on compatible mitochondrial DNA and reprogramming factors

Interspecies somatic cell nuclear transfer (iSCNT) involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed oocytes can be cultured in vitro to blastocyst, the final stage of preimplantation development. However, they often arrest during the early stages of preimplantation development; fail to reprogramme the somatic nucleus; and eliminate the accompanying donor cell's mitochondrial DNA (mtDNA) in favour of the recipient oocyte's genetically more divergent population. This last point has consequences for the production of ATP by the electron transfer chain, which is encoded by nuclear and mtDNA. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility on successful development. Initially, we transferred murine fetal fibroblasts into enucleated porcine oocytes, which resulted in extremely low blastocyst rates (0.48%); and failure to replicate nuclear DNA and express Oct-4, the key marker of reprogramming. Using allele specific-PCR, we detected peak levels of murine mtDNA at 0.14±0.055% of total mtDNA at the 2-cell embryo stage and then at ever-decreasing levels to the blastocyst stage (<0.001%). Furthermore, these embryos had an overall mtDNA profile similar to porcine embryos. We then depleted porcine oocytes of their mtDNA using 10 μM 2',3'-dideoxycytidine and transferred murine somatic cells along with murine embryonic stem cell extract, which expressed key pluripotent genes associated with reprogramming and contained mitochondria, into these oocytes. Blastocyst rates increased significantly (3.38%) compared to embryos generated from non-supplemented oocytes (P<0.01). They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout early preimplantation development. At later stages, these embryos possessed 49.99±2.97% murine mtDNA. They also exhibited an mtDNA profile similar to murine preimplantation embryos. Overall, these data demonstrate that the addition of species compatible mtDNA and reprogramming factors improves developmental outcomes for iSCNT embryos.     

Quantification of mtDNA in single oocytes, polar bodies and subcellular components by real-time rapid cycle fluorescence monitored PCR

Oocytes, in general, are greatly enriched in mitochondria to support higher rates of macromolecular synthesis and critical physiological processes characteristic of early development. An inability of these organelles to amplify and/or to accumulate ATP has been linked to developmental abnormality or arrest. The number of mitochondrial genomes present in mature mouse and human metaphase II oocytes was estimated by fluorescent rapid cycle DNA amplification, which is a highly sensitive technique ideally suited to quantitative mitochondrial DNA (mtDNA) analysis in individual cells. A considerable degree of variability was observed between individual samples. An overall average of 1.59 x 10(5) and 3.14 x 10(5) mtDNA molecules were detected per mouse and human oocyte, respectively. Furthermore, the mtDNA copy number was examined in polar bodies and contrasted with the concentration in their corresponding oocytes. In addition, the density of mtDNA in a cytoplasmic sample was estimated in an attempt to determine the approximate number of mitochondria transferred during clinical cytoplasmic donation procedures as well as to develop a clinical tool for the assessment and selection of oocytes during in vitro fertilisation procedures. However, no correlation was identified between the mtDNA concentration in either polar bodies or cytoplasmic samples and their corresponding oocyte.     

Phase Transition Temperature and Chilling Sensitivity of Bovine Oocytes

A limiting factor for achieving cryopreservation of oocytes is direct chilling injury (DCI), which occurs during cooling. DCI, or cold shock, is defined as an irreversible damage expressed shortly after exposure to low, but not freezing, temperatures. The primary target of DCI is thought to be the plasma membrane. Recently, an association between DCI in sperm and the thermotropic phase transition of their membrane lipids was demonstrated. In the present study, we examined the phase transition of the membrane lipids of immature andin vitro-matured bovine oocytes during cooling, using Fourier transform infrared spectroscopy (FTIR). The phase transition of the membrane lipids of oocytes at the germinal vesicle (GV) stage occurred between 13 and 20°C, while a very broad phase transition, which centered around 10°C, was observed for mature oocytes (MII) stage. Thermotropic phase transitions were demonstrated to be related to the temperature at which DCI affected the integrity of the oocyte membranes. When immature oocytes were cooled to 13°C, fewer oocytes (40%) retained their membrane integrity than after exposure to 4°C (51%) or holding them at 38°C (78%), (as determined by the Fluorescein Diacetate-FDA test). This finding might suggest that holding immature oocytes at the phase transition temperature is more damaging to their membranes than exposure to lower temperatures. By contrast, no significant differences in membrane integrity were observed whenin vitro-matured oocytes were cooled to the same temperatures. Subsequently, GV oocytes were cooled to 4°C, and 26% underwent maturation and 19% underwent fertilizationin vitro. In vitro-matured oocytes that were cooled to 4°C displayed a slightly decreased rate of fertilization; the overall fertilization was 60% with 24% polyspermy, rather than the 76% fertilization rate with 12% polyspermy obtained with those not subjected to cooling. The high rate of polyspermy indicates that a site(s) other than the plasma membrane is affected during cooling of bovine oocytes. Nucleated bovine GV oocytes were electrofused within vitro-matured and enucleated oocytes, and then cooled to 4°C. Evaluation of the membrane integrity of the fused oocytes showed that these oocytes are chilling resistant, which strongly suggests that alteration of the membrane composition of an oocyte can change the cell's susceptibility to low temperatures. This finding led to an improvement in the survival of oocytes after cryopreservation.