Biomedical subjects

V Zakhartchenko

Publications and source records attributed to V Zakhartchenko.

29 records · Page 2Linked to original sources

PubMed1999

Coenzyme Q(10) in submicron-sized dispersion improves development, hatching, cell proliferation, and adenosine triphosphate content of in vitro-produced bovine embryos.

Coenzyme Q(10) (CoQ(10)) is an essential component of the plasma membrane ion transporter (PMIT) system and of the electron transport chain in the inner mitochondrial membrane. Because of its intrinsic functions in cell growth and energy metabolism (ATP synthesis), and its protective effects against oxidative stress, CoQ(10) is a good candidate for supporting growth of cells in culture. However, because of its quinone structure, CoQ(10) is extremely lipophilic and practically insoluble in water. We used a specific technology to prepare a submicron-sized dispersion of CoQ(10), inhibiting re-crystallization by a stabilizer. This dispersion, which exhibits a very large specific surface area for drug dissolution, was tested as a supplement for the in vitro culture of bovine embryos in a chemically defined system. The rate of early cleavage of embryos (5- to 8-cell stages) was evaluated 66 h postinsemination (hpi) and was highest in medium supplemented with 30 or 100 microM CoQ(10) (66.5 +/- 0.8% and 68.7 +/- 1.1%, respectively) and lowest in 10 microM CoQ(10) (55.3 +/- 0.8%). The proportions of oocytes developing to blastocysts by 186 hpi were 19.0 +/- 0.6% and 25.2 +/- 0.3% in medium supplemented with 10 microM and 30 microM CoQ(10), respectively, and were significantly (p < 0.001) higher than those obtained with the equivalent amounts of stabilizer (9.9 +/- 0.4% and 11.3 +/- 0.4%). In the presence of 30 microM CoQ(10), significantly (p < 0.001) more blastocysts hatched by 210 hpi than in the equivalent amount of stabilizer (31.8 +/- 1.3 vs. 8.4 +/- 2.2). Expanded blastocysts produced in the presence of 30 microM CoQ(10) had significantly (p < 0.01) more inner cell mass cells and trophectoderm cells, and a significantly (p < 0.001) increased ATP content as compared to expanded blastocysts produced in the presence of the corresponding amount of stabilizer. Our results show that noncrystalline CoQ(10) in submicron-sized dispersion supports the development and viability of bovine embryos produced in a chemically defined culture system.

Adenosine Triphosphate↗

PubMed1999

Effects of serum starvation and re-cloning on the efficiency of nuclear transfer using bovine fetal fibroblasts.

The developmental potential of bovine fetal fibroblasts was evaluated using nuclear transfer. Fibroblasts from a 37-day-old fetus were fused to enucleated oocytes before activation. Nuclei of starved (cultured for 8 days in medium containing 0.5% serum) fibroblasts supported the development of reconstructed embryos to the blastocyst stage significantly better than those of non-starved fibroblasts (39% versus 20%; P < 0.05). When nuclear transfer morulae derived from starved or non-starved fibroblasts were used for re-cloning, the proportion of blastocysts (52 and 55%, respectively) obtained with these embryonic nuclei was significantly higher than it was with fibroblast nuclei used in the first round of nuclear transfer (P < 0.05 and P < 0.001, respectively). After transfer of blastocysts derived from non-starved and starved fibroblasts, respectively, 33% (1/3) and 78% (7/9) of recipients were pregnant on day 30 as assessed by ultrasonography. On day 90, the corresponding pregnancy rates were 33% (1/3) and 63% (5/8). Two live male twin calves, derived from non-starved fibroblasts, were delivered by Caesarean section at day 281 of gestation. This study demonstrates a positive effect of serum starvation on the efficiency of nuclear transfer using bovine fetal fibroblasts. The efficiency of nuclear transfer could be further increased by recloning.

Animals↗

PubMed1998

Nuclear transfer in mammals: recent developments and future perspectives.

A clone can be defined as a set of genetically identical animals. Small clones of two or occasionally up to four identical animals can be obtained by embryo splitting or blastomere separation. Embryo cloning by nuclear transfer involves the transfer of genetic material from a donor cell (karyoplast) to the cytoplasm of an oocyte or zygote from which the genetic material has been removed (cytoplast). In farm animals, metaphase II oocytes are most widely used as cytoplasts. There are now many factors known to influence the efficiency of embryo cloning by nuclear transfer. These include stage of development and cell cycle of donor cells, the choice of the recipient cell, the methods for activation of oocytes, the cell cycle coordination between donor cell and recipient cytoplast, and the method for fusion between nuclear donor and recipient cytoplast. Recent progress in cloning embryos and animals from cultured cells of embryonic, fetal, or adult origin offers a wide spectrum of potential applications of nuclear transfer, such as the unlimited multiplication of elite embryos or animals from selected matings and the potential for precise genetic modification of farm animals for gene farming or xenotransplantation.

Animals↗

PubMed1998

Composition of parental mitochondrial DNA in cloned bovine embryos.

We have investigated parental mitochondrial DNA (mtDNA) in cloned bovine embryos obtained by intraspecific cytoplast-blastomere fusion. Analysis of two-cell to blastocyst stage embryos revealed that in contrast to the exclusion of paternal (sperm) mtDNA during sexual inheritance in the cytoplast-blastomere fusion complexes, there was mixing and co-existence of parental mtDNA. The mixing of mtDNA was non-balanced with the minority deriving from the blastomere. The constant content of mtDNA during embryogenesis until the blastocyst stage suggesting an absence of mtDNA replication was shown for conventional 'in vitro fertilised' (IVF) embryos and for cloned embryos. The ratio of parental mtDNA was in accordance with the estimated quantitative participation of mtDNA from the fusion partners.

Animals↗

PubMed1998

Non-balanced mix of mitochondrial DNA in cloned cattle produced by cytoplast-blastomere fusion.

We have investigated the transmission of parental mitochondrial DNA (mtDNA) in three clones of born cattle obtained by intraspecific cytoplast-blastomere fusion. Using allele-specific TaqMan PCR a low level transmission of blastomere mtDNA (DB mtDNA) into the cloned offspring was detected, thereby generating a heteroplasmic population of mtDNA. The amount of DB mtDNA was 13% and 18% in two animals of a clone which derived from a 24-cell morula and 0.6% and 0.4% in two calves of clonal origin derived from a 92-cell morula. These values are in accordance with the tendency expected for neutral mtDNA segregation that the fewer cell divisions that have occurred in the donor embryo, the higher the amount of DB mtDNA. We also found a strong decrease of DB mtDNA which was about three orders of magnitude in the third clone derived from a 52-cell morula stage.

Animals↗

PubMed1998

A reliable procedure for differential staining of in vitro produced bovine blastocysts: comparison of tissue culture medium 199 and Ménézo's B2 medium.

A reliable double-dye technique has been established for counting the number of inner cell mass and trophectoderm cells of in vitro produced bovine blastocysts. The latter were first incubated in a 1:2 dilution of a rabbit antiserum raised against a mixture of recombinant bovine interferon tau and serum containing medium conditioned by in vitro produced trophoblastic vesicles for 45 min at 39 degrees C. Subsequently, the blastocysts were incubated in a 5% (v/v) solution of guinea pig complement in phosphate-buffered saline containing 50 micrograms/ml propidium iodide for 45 min at 39 degrees C. Then the blastocysts were transferred to ice-cold absolute ethanol containing 25 micrograms/ml bisbenzimide and evaluated under a fluorescence microscope. Since trophectoderm cells were permeabilised by antibody-mediated complement lysis, they were stained by propidium iodide (red or pink). Bisbenzimide can enter lysed and non-lysed cells and therefore stained also inner cell mass cells (blue) which had been protected from complement lysis by trophectoderm cells. This modified procedure proved to be very reliable for differential cell staining of bovine blastocysts produced under various culture conditions. A comparison of blastocysts produced in Ménézo's B2 vs. TCM 199 media (both supplemented with 10% serum from cows at oestrus) revealed significant (P < 0.01) differences in total cell numbers (119 +/- 24 vs. 84 +/- 10; mean +/- SD) and in the numbers of trophectoderm cells (79 +/- 19 vs. 57 +/- 8) and inner cells mass cells (40 +/- 7 vs. 26 +/- 5) between the two culture systems. The modified staining procedure presented here is a valuable tool for evaluating the quality of in vitro produced bovine blastocysts and for improving of culture conditions.

Animals↗

PubMed1997

Karyoplast-cytoplast volume ratio in bovine nuclear transfer embryos: effect on developmental potential.

To evaluate the effect of karyoplast-cytoplast ratio on the development of nuclear transfer embryos, karyoplasts from day 4, day 5, and day 6 embryos were transferred to oocytes enucleated with different volumes of cytoplasm: Type 1, removal of a small volume of cytoplasm equivalent to the first polar body, Type 2, removal of a volume of cytoplasm approximately equal to the volume of the respective karyoplast, and Type 3, removal of half of the oocyte volume. In addition, the effect of experimental reduction of karyoplast cytoplasm was investigated in day 4 and day 5 karyoplasts. Intact day 4 karyoplasts fused to Type 3 cytoplasts did not support development to blastocysts, whereas these karyoplasts yielded blastocysts in combination with Type 1 (7%) and Type 2 cytoplasts (12%). After experimental reduction of cytoplasmic volume in day 4 karyoplasts, blastocysts (10%) were also obtained after fusion with Type 3 cytoplasts, probably due to reduction of cytoplasmic chimerism. With day 5 karyoplasts, blastocyst rate was higher in combination with Type 2 (34%) than with Type 1 (19%) and Type 3 cytoplasts (16%; P < 0.05). The use of day 6 intact karyoplasts resulted in a significantly (P < 0.05) higher proportion of blastocysts when fused with Type 2 (38%) or Type 1 cytoplasts (34%) than with Type 3 cytoplasts (16%). These results suggest that enucleation of oocytes with a volume similar to that of the respective karyoplast creates better conditions for cell cycle interactions with all types of karyoplasts than enucleation with minimal or large volume of cytoplasm.

Animals↗

PubMed1997

Support for the development of bovine embryos in vitro by secretions of bovine trophoblastic vesicles derived in vitro.

This study evaluated whether trophoblastic tissue derived in vitro secretes factors that support bovine embryonic development in vitro. The embryotrophic activity of these secretions was analysed in three different culture conditions based on TCM-199: (1) in a routine culture system using cumulus cells and 10% oestrous cow serum; (2) without cells but with 10% oestrous cow serum; and (3) under serum-free conditions. Rates of development to the 5-8-cell and blastocyst stages, as well as numbers of inner cell mass and trophectoderm cells of blastocysts were determined. In the absence of cumulus cells, cleavage rates of 5-8-cell embryos were significantly (P < 0.05) higher in trophoblastic vesicle-conditioned medium than in TCM-199 in both the presence (71% versus 49%) and absence (70% versus 49%) of serum. Trophoblastic vesicle-conditioned medium had a significant (P < 0.05) positive effect on the rate of development to the blastocyst stage when compared with TCM-199 in the presence of cumulus cells and serum (39% versus 33%), only serum (26% versus 19%), or in the absence of cells and serum (21% versus 5%). The numbers of inner cell mass and trophectoderm cells, and total number of cells in blastocysts produced in the cumulus cell coculture system in serum-free trophoblastic vesicle-conditioned medium or TCM-199 supplemented with serum were greater than those of blastocysts produced without cumulus cells or serum. Fractionation of serum-free trophoblastic vesicle-conditioned medium by ultrafiltration (10 kDa cut off) confined the embryotrophic activity mainly to the low molecular mass fraction. This study shows that serum-free trophoblastic vesicle-conditioned medium contains potent embryotrophic factors which act in a complementary manner to those secreted by cumulus cells and those supplemented with serum and result in reliably high blastocyst rates in the range of 40%. Since contamination of trophoblastic vesicle-conditioned medium with serum proteins can be avoided, this medium may be a reasonable source for the purification of specific embryotrophic factors.

Animals↗

PubMed1996

Nuclear transfer in cattle using in vivo-derived vs. in vitro-produced donor embryos: effect of developmental stage.

To determine the best developmental stage of donor embryos for yielding the highest number of clones per embryo, we compared the efficiencies of nuclear transfer when using blastomeres from morulae or morulae at cavitation, or when using inner-cell-mass cells of blastocysts as nuclear donors. This comparison was done both on in vivo-derived and in vitro-produced donor embryos. In experiment 1, with in vivo-derived donor embryos, nuclei from morulae at cavitation supported the development of nuclear transfer embryos to the blastocyst stage (36%) at a rate similar to that of nuclei from morulae (27%), blastomeres from morulae at cavitation being superior (P < 0.05) to inner-cell-mass cells from blastocysts (21%). The number of blastocysts per donor embryo was significantly (P < 0.05) higher when using nuclei from morulae at cavitation (15.7 +/- 4.1) rather than nuclei from morulae (9.8 +/- 5.5) or blastocysts (6.3 +/- 3.3). With in vitro-produced donor embryos (experiment 2), nuclei from morulae yielded slightly more blastocysts (32%) than nuclei from morulae at cavitation (29%), both stages being superior to nuclei from blastocysts (15% development to the blastocyst stage). Morulae at cavitation yielded a higher number of cloned blastocysts per donor embryo (11.5 +/- 5.9) than did morulae (9.3 +/- 3.2) and blastocysts (3.3 +/- 1.4). Transfer of cloned embryos originating from in vivo-derived morulae, morulae at cavitation, and blastocysts resulted in four pregnancies (10%), three pregnancies (7%), and one (17%) pregnancy on day 45. The corresponding numbers of calves born were 3 (4%), 3 (7%), and 0, respectively. After transfer of blastocysts derived from in vitro nuclear donor morulae (n = 16) and morulae at cavitation (n = 7), two (20%) and two (50%) recipients, respectively, were pregnant on day 45. However, transfer of seven cloned embryos from in vitro donor blastocysts to three recipients did not result in a pregnancy. Using in vitro-produced donor embryos, calves were only obtained from morula-stage donors (13%). Our results indicate that the developmental stage of donor embryos affects the efficiency of nuclear transfer, with morulae at cavitation yielding a high number of cloned blastocysts.

Animals↗

PubMed1995

Effect of donor embryo cell number and cell size on the efficiency of bovine embryo cloning.

To establish reliable criteria for the evaluation of nuclear donor embryos, we studied the effect of cell number and cell size of in vitro produced day 6 donor morulae on the rate of blastocyst formation following nuclear transfer to in vitro matured oocytes. In experiment 1, donor embryos were divided into three groups with low (25-34), intermediate (40-55), and high (60-81) blastomere numbers. Transfer of nuclei from day 6 morulae with intermediate and high cell numbers resulted in a significantly higher blastocyst rate (31% and 32%, respectively) than use of nuclei from day 6 morulae with low cell numbers (17%) or nuclei from day 7 morulae with 50-83 blastomeres (19%). This suggests that blastomeres from the developmentally advanced day 6 morulae are more viable than blastomeres from retarded embryos. In experiment 2, we evaluated the effect of blastomere size in day 6 donor morulae with intermediate (40-55) or high (60-81) cell numbers on the efficiency of nuclear transfer. In both classes of embryos, small blastomeres were better nuclear donors than large blastomeres. The rates of development to the blastocyst stage were 28% versus 15% (40-55 cells) and 41% versus 25% (60-81 cells), suggesting that small blastomeres include a higher proportion of totipotent cells than the polarized large blastomeres. Our results demonstrate that blastomere number and size markedly affect the efficiency of nuclear transfer and therefore are useful criteria for evaluating nuclear donor embryos. These parameters are easy to determine and may therefore be helpful to improve the efficiency of cattle cloning.

Animals↗

PubMedDate not supplied

Effect of donor cell age on the efficiency of nuclear transfer in rabbits.

The ability of rabbit fibroblasts of different ages to be reprogrammed following nuclear transfer (NT) to aged recipient oocytes was evaluated. The rate of NT blastocysts reconstructed with presumptive G1 stage morula cells or fetal fibroblasts was significantly higher (41.5% and 51.4%) than was those of cloned embryos reconstructed with fibroblasts from young (4-month-old) or aged (5-year-old) animals (16.7% and 7.1%, respectively, P < 0.025). Serum starvation significantly increased the development of NT embryos to the morula-blastocyst stage (67.6% versus 22.9%, P < 0.025). Transfer of 168 NT embryos derived from nuclei of morula cells and 106 control embryos into 21 recipients resulted in 10 pregnancies, 2 NT and 18 control pups, respectively. In the first experiment, transfer of 142 cleaved NT embryos reconstructed with fetal fibroblasts and 86 control embryos into eight recipient does resulted in five pregnancies and the birth of 20 control pups. In the second experiment, after transfer of 112 NT embryos derived from fetal fibroblasts into six recipients, 10 (8.9%) sites of implantation were revealed in two does (33.3%) on day 14 of gestation. This study provides evidence that nuclei of morula cells and fetal and adult fibroblasts differ in their ability to be reprogrammed by recipient cytoplasm following nuclear transfer.

Animals↗