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1.
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.  相似文献   

2.
Mitochondrial metabolic capacity and DNA replication have both been shown to affect oocyte quality, but it is unclear which one is more critical. In this study, immature oocytes were treated with FCCP or ddC to independently inhibit the respective mitochondrial metabolic capacity or DNA replication of oocytes during in vitro maturation. To differentiate their roles, we evaluated various parameters related to oocyte maturation (germinal vesicle break down and nuclear maturation), quality (spindle formation, chromosome alignment, and mitochondrial distribution pattern), fertilization capability, and subsequent embryo developmental competence (blastocyst formation and cell number of blastocyst). Inhibition of mitochondrial metabolic capacity with FCCP resulted in a reduced percent of oocytes with nuclear maturation; normal spindle formation and chromosome alignment; evenly distributed mitochondria; and an ability to form blastocysts. Inhibition of mtDNA replication with ddC has no detectable effect on oocyte maturation and mitochondrial distribution, although high-dose ddC increased the percent of oocytes showing abnormal spindle formation and chromosome alignment. ddC did, however, reduce blastocyst formation significantly. Neither FCCP nor ddC exposure had an effect on the rate of fertilization. These findings suggest that the effects associated with lower mitochondrial DNA copy number do not coincide with the effects seen with reduced mitochondrial metabolic activity in oocytes. Inhibiting mitochondrial metabolic activity during oocyte maturation has a negative impact on oocyte maturation and subsequent embryo developmental competence. A reduction in mitochondrial DNA copy number, on the other hand, mainly affects embryonic development potential, but has little effect on oocyte maturation and in vitro fertilization.  相似文献   

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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.  相似文献   

5.
The aim of this study was to examine the effect of different stimulation protocols on oocyte granularity and to determine the influence of cytoplasmic granularity on further embryo development. A total of 2448 oocytes from 393 intracytoplasmic sperm injection (ICSI) cycles were analysed retrospectively. Oocytes were classified into 5 groups according to cytoplasmic granularity. (A) no granule or 1-2 small (<5 μm) granules; (B) more than 3 small granules; (C) large granules (>5 μm); (D) refractile body; (E) dense centrally located granular area. Correlation between characteristics of hormonal stimulation, oocyte granularity and embryo development was analysed. The occurrence of cytoplasmic granularity was influenced by the patient's age and characteristics of stimulation. The type of granulation had no effect on fertilization rate and zygote morphology. However, some type of granulation resulted in a lower cleavage rate and more fragmented embryos. Our results provided additional information on how hormonal stimulation affects oocyte quality. While cytoplasmic granularity seems not to have an effect on fertilization and embryo development, the presence of refractile body in the oocyte is associated with reduced cleavage rates and impaired embryo development.  相似文献   

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Maternal diabetes has been demonstrated to adversely affect preimplantation embryo development and pregnancy outcomes. Emerging evidence has implicated that these effects are associated with compromised oocyte competence. Several developmental defects during oocyte maturation in diabetic mice have been reported over past decades. Most recently, we further identified the structural, spatial and metabolic dysfunction of mitochondria in oocytes from diabetic mice, suggesting the impaired oocyte quality. These defects in the oocyte may be maternally transmitted to the embryo and then manifested later as developmental abnormalities in preimplantation embryo, congenital malformations, and even metabolic disease in the offspring. In this paper, we briefly review the effects of maternal diabetes on oocyte quality, with a particular emphasis on the mitochondrial dysfunction. The possible connection between dysfunctional oocyte mitochondria and reproductive failure of diabetic females, and the mechanism(s) by which maternal diabetes exerts its effects on the oocyte are also discussed.  相似文献   

9.
ICSI and embryo biopsy are routine methods used for assisted reproduction. However, their impact on embryo quality is still poor studied. Moreover, oocyte size is also a crucial factor for blastocyst production. In this study effect of oocyte size, ICSI and embryo biopsy was assessed in terms of incidence of apoptosis and blastocyst development. IVM-oocytes from prepubertal goats were fertilized by ICSI or IVF. Embryos obtained were divided depending on oocyte size, biopsied at day-4 post-insemination/injection and cultured for additional 4-5 days. Apoptotic cell number was assessed by TUNEL staining in day-4 embryos and blastocysts obtained. In each diameter group, ICSI did not affect embryo development, blastocyst cell number and embryo apoptotic grade in comparison to IVF. Embryo biopsy did not affect blastocyst rate and apoptotic cell number, but decreased blastocyst cell number (P=0.0018). Moreover, there was a negative relationship between blastocyst cell number and apoptotic grade (P<0.05). In conclusion, ICSI and embryo biopsy do not have negative effect on embryo quality and development. However, oocyte size has a positive relationship on blastocyst yield and quality.  相似文献   

10.
It has been shown recently that there is premature mitochondria biosynthesis in blastocysts from older women whose egg or embryo quality is poor and that aneuploid blastocysts also have a high number of mitochondrial DNA (mtDNA) copies. Whether nondiploidy/aneuploidy or reduced egg or embryo quality causes premature mitochondrial biosynthesis is not known. This study constructed haploid, diploid, triploid, and tetraploid blastocysts by parthenogenetic activation, intracytoplasmic sperm injection with one or two sperm heads, blastomere electrofusion, respectively, and generated reduced cytoplasm quality embryos from diabetic mouse and in vitro fertilization of aged oocytes, and examined whether nondiploidy or reduced cytoplasm quality causes premature mitochondrial biosynthesis. MtDNA numbers of each blastocyst from different models were tested by absolute quantitative real-time polymerase chain reaction. It was found that mtDNA content in preimplantation embryos was not associated with their chromosome ploidy, while mtDNA copy numbers in embryos with suboptimal quality were increased. Therefore, it might be the reduced cytoplasmic quality, and not chromosome nondiploidy, that causes premature mitochondria biosynthesis in blastocysts.  相似文献   

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Mitochondria isolated from stage 3 (about half-grown) oocytes of Xenopus laevis exhibit a DNA synthetic rate in vitro of 2.35 ± 0.28 pg/oocyte/h. Similarly, stage 6 (full-grown) oocyte mitochondria synthesize DNA (mtDNA) at 0.28 ± 0.02 pg/oocyte/h. By comparison, the rate of mtDNA synthesis by intact stage 6 oocytes following microinjection of [3H]-dTTP was calculated to be 0.43 ± 0.08 pg/oocyte/h, indicating that the observed in vitro rates may represent minimum values. Measurements of DNA polymerase activity associated with mitochondria isolated from stage 3 oocytes are almost three times those recorded with stage 6 oocyte mitochondria. It appears that active replication of complete mtDNA molecules, which accompanies accumulation of mitochondria by the egg, is terminated midway through oogenesis.  相似文献   

13.
Because the mtDNA amount remains stable in the early embryo until uterine implantation, early human development is completely dependent on the mtDNA pool of the mature oocyte. Both quantitative and qualitative mtDNA defects therefore may negatively impact oocyte competence or early embryonic development. However, nothing is known about segregation of mutant and wild-type mtDNA molecules during human meiosis. To investigate this point, we compared the mutant levels in 51 first polar bodies (PBs) and their counterpart (oocytes, blastomeres, or whole embryos), at risk of having (1) the "MELAS" m.3243A>G mutation in MT-TL1 (n = 30), (2) the "MERRF" m.8344A>G mutation in MT-TK (n = 15), and (3) the m.9185T>G mutation located in MT-ATP6 (n = 6). Seven out of 51 of the PBs were mutation free and had homoplasmic wild-type counterparts. In the heteroplasmic PBs, measurement of the mutant load was a rough estimate of the counterpart mutation level (R(2) = 0.52), and high mutant-load differentials between the two populations were occasionally observed (ranging from -34% to +34%). The mutant-load differentials between the PB and its counterpart were higher in highly mutated PBs, suggestive of a selection process acting against highly mutated cells during gametogenesis or early embryonic development. Finally, individual discrepancies in mutant loads between PBs and their counterparts make PB-based preconception diagnosis unreliable for the prevention of mtDNA disorder transmission. Such differences were not observed in animal models, and they emphasize the need to conduct thorough studies on mtDNA segregation in humans.  相似文献   

14.
The role of mitochondria as a nexus of developmental regulation in mammalian oogenesis and early embryogenesis is emerging from basic research in model species and from clinical studies in infertility treatments that require in vitro fertilization and embryo culture. Here, mitochondrial bioenergetic activities and roles in calcium homeostasis, regulation of cytoplasmic redox state, and signal transduction are discussed with respect to outcome in general, and as possible etiologies of chromosomal defects, maturation and fertilization failure in human oocytes, and as causative factors in early human embryo demise. At present, the ability of mitochondria to balance ATP supply and demand is considered the most critical factor with respect to fertilization competence for the oocyte and developmental competence for the embryo. mtDNA copy number, the timing of mtDNA replication during oocyte maturation, and the numerical size of the mitochondrial complement in the oocyte are evaluated with respect to their relative contribution to the establishment of developmental competence. Rather than net cytoplasmic bioenergetic capacity, the notion of functional compartmentalization of mitochondria is presented as a means by which ATP may be differentially supplied and localized within the cytoplasm by virtue of stage-specific changes in mitochondrial density and potential (ΔΨm). Abnormal patterns of calcium release and sequestration detected at fertilization in the human appear to have coincident effects on levels of mitochondrial ATP generation. These aberrations are not uncommon in oocytes obtained after ovarian hyperstimulation for in vitro fertilization. The possibility that defects in mitochondrial calcium regulation or bioenergetic homeostasis could have negative downstream development consequences, including imprinting disorders, is discussed in the context of signaling pathways and cytoplasmic redox state.  相似文献   

15.
Unsatisfactory reproductive performance in dairy cows, such as reduced conception rates, in addition to an increased incidence of early embryonic mortality, is reported worldwide and has been associated with a period of negative energy balance (NEB) early post partum. Typically, NEB is associated with biochemical changes such as high non-esterified fatty acid (NEFA), high β-hydroxybutyrate (β-OHB) and low glucose concentrations. The concentrations of these and other metabolites in the follicular fluid (FF) of high-yielding dairy cows during NEB were determined and extensively analyzed, and then were replicated in in vitro maturation models to investigate their effect on oocyte quality. The results showed that typical metabolic changes during NEB are well reflected in the FF of the dominant follicle. However, the oocyte seems to be relatively isolated from extremely elevated NEFA or very low glucose concentrations in the blood. Nevertheless, the in vitro maturation models revealed that NEB-associated high NEFA and low glucose levels in the FF are indeed toxic to the oocyte, resulting in deficient oocyte maturation and developmental competence. Induced apoptosis and necrosis in the cumulus cells was particularly obvious. Furthermore, maturation in saturated free fatty acid-rich media had a carry-over effect on embryo quality, leading to reduced cryotolerance of day 7 embryos. Only β-OHB showed an additive toxic effect in moderately hypoglycemic maturation conditions. These in vitro maturation models, based on in vivo observations, suggest that a period of NEB may hamper the fertility of high-yielding dairy cows through increased NEFA and decreased glucose concentrations in the FF directly affecting oocyte quality. In addition to oocyte quality, these results also demonstrate that embryo quality is reduced following an NEB episode. This important observation may be linked to the typical diet provided to stimulate milk yield, or to physiological adaptations sustaining the high milk production. Research into this phenomenon is ongoing.  相似文献   

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17.
Promoting lipid utilization with l-carnitine to improve oocyte quality   总被引:1,自引:0,他引:1  
Successful embryo and fetal development is dependent on the quality of the oocyte from which it was derived. Several studies to date have demonstrated the link between appropriate metabolism and sufficient ATP production with oocyte quality and preimplantation embryo development. Metabolism of fatty acids for the purpose of synthesizing ATP occurs within mitochondria via β-oxidation and entry of fatty acids into this organelle is the rate-limiting step in this process. Transport of activated fatty acids into mitochondria is catalyzed by carnitine palmitoyl transferase-I (CPTI) which also requires the metabolite carnitine. Once inside the mitochondrial matrix, fatty acids are broken down into acetyl CoA molecules which are further metabolized via the TCA cycle and electron transport chain to produce ATP. The potential to improve oocyte quality by modulating fatty acid metabolism and β-oxidation with carnitine in culture media formulations or via dietary supplementation has received little attention. This review summarizes studies to date investigating the developmental importance of β-oxidation through the use of metabolic inhibitors and whether regulation by carnitine, in vitro or in vivo, has beneficial effects on oocyte and embryo development. Overall, there is little evidence to date that dietary carnitine can improve oocyte quality or female fertility; however inclusion of l-carnitine to in vitro oocyte maturation and embryo growth media improves embryo outcomes, most likely by supplying the oocyte and embryo with an essential co-factor required to utilize fatty acids.  相似文献   

18.
Oocyte cytoplasm plays a prominent role in cloned embryonic development. To investigate the influence of oocyte cytoplasmic amount on cloned embryo development, we generated bovine somatic cell nuclear transfer (SCNT) embryos containing high (30-40% of the cytoplasm was removed), medium (15-25% of the cytoplasm was removed) and low (<10% of the cytoplasm was removed) nucleocytoplasmic volume ratios (N/C) using enucleated metaphase II oocyte as recipient, and fibroblast as donor nucleus, and analyzed the expression levels of ND1, Cytb and ATPase6, as well as the embryonic quality. The results indicated: (1) the process of embryonic development was not influenced by <40% of cytoplasm removal; (2) the rate of blastocyst formation, the total number of blastomere and the ratio of ICM to TE were inversely proportional to the N/C; (3) SCNT embryos with reduced volume equal to 75-85% or >90% of an intact oocyte volume showed similar karyotype structure of the donor cells; (4) the number of mtDNA copy was larger in low N/C embryos than that in medium or high N/C embryos, and the expression levels of each gene hardly varied from the 2-cell to 8-cell stage, while the expression levels increased dramatically at the blastocyst stage; (5) from 16-cell to the blastocyst stage, the change of the expression level of each gene was not significant between low N/C embryos and IVF embryos, but it was more significant than those of high or medium N/C embryos. The results suggest that the decrease of mtDNA copy number and mitochondrial gene expression may be related to the impairment in early embryonic development, and removal of <10% adjacent cytoplasm volume may be optimal for bovine SCNT embryo development.  相似文献   

19.
Mitochondrial dysfunction in reproduction   总被引:5,自引:0,他引:5  
Jansen RP  Burton GJ 《Mitochondrion》2004,4(5-6):577-600
The mitochondrial genome passes from one generation to the next by way of the egg's cytoplasm, so ordinarily an individual's mitochondrial DNA (mtDNA) is entirely derived from his or her mother. A potential mother has a finite number of eggs, or oocytes, all of which were formed when she herself was still a fetus, many years before she can conceive. The eggs are progressively depleted through childhood and her reproductive years at a much faster rate than is accounted for by ovulation. Up to a decade before the ultimate depletion of ovarian follicles (and hence oocytes) at or soon after menopause, cytoplasmic senility of the remaining eggs leads to physiological sterility; a phenomenon that is suspected of being mitochondrially based and has been termed the oopause. When ovulation and conception occur, oxidative phosphorylation and other mitochondrial functions of the fertilized oocyte are thought to be essential to the early embryo well before it implants in the uterus. The competition between follicles to deliver the oocyte that will be fertilized and which will found a new generation could also be mitochondrially based, but the mechanism remains to be elucidated. Increasing experience with the culture of human embryos in vitro is highlighting the importance of mitochondrial metabolism generally, and the avoidance of excessive generation of reactive oxygen species in particular. Paradoxes abound in the experimental data, however. Although natural selection operates on mitochondria only in females (and in extreme cases through the survival of their offspring), reproductive disturbance from mitochondrial mutations is most obvious in males, who typically have reduced sperm motility. mtDNA point mutations such as T8993G, which is serious enough to cause the death of infants from Leigh disease in the first few years of life, can carry through the female germ line apparently unhindered; yet mtDNA deletions that cause a less severe phenotype, and which typically manifest at a later age, are effectively blocked from transmission to offspring--a phenomenon in accord with early experimental observations that deleted mtDNA species are less common in cleaving embryos than in unselected preovulatory oocytes. A mitochondrial basis for ooplasmic aging has not been convincingly established, but the novel IVF-based practice of micro-aspiration and transfer of ooplasm from younger eggs to older eggs, which includes the transfer of mitochondria, appears in preliminary studies to have some clinical efficacy in rejuvenating fertility in older women.  相似文献   

20.
Nutrition of the donor cow can influence oocyte and embryo quality, which can affect the success of embryo transfer. Severe undernutrition compromised ovarian follicular development, with implications for superovulatory response and embryo quality. In postpartum lactating cows, undernutrition or inability to consume sufficient nutrients delayed resumption of ovulation, reduced the number of follicles, and compromised oocyte quality. Moderate undernutrition of nonlactating cows was unlikely to affect embryo quality; conversely, nonlactating animals on maintenance diets usually had better superovulatory responses and improved oocyte competence and embryo quality. The negative effects of overfeeding are thought to be mediated by alterations in endocrine cues, such as hyperinsulinemia and increased glucose and IGF-I, which may interfere with glucose transport in the embryo and increase apoptosis. Manipulating energy sources such as carbohydrates and fatty acids (FA) may influence embryo viability, but the effects of FA were not consistent in vitro; increasing concentrations of unsaturated FA in follicular and embryonic cells usually improved embryo viability and resistance to cryopreservation. Excess protein intake and increased urea and ammonia in body fluids can be toxic to embryos, impairing their development; these effects seemed to be associated with alterations in uterine pH and granulosa cell function. Likewise, toxins in feeds (e.g. gossypol), reduced embryo development and increased pregnancy losses. Diet of donor cows should be formulated to optimize the supply of nutrients to meet needs; however, manipulating energy intake, source of FA and protein content of donor diets, particularly moderate underfeeding in nonlactating cows, may further optimize responses.  相似文献   

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