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ABSTRACT: The removal of chromosomes from recipient oocytes is one of the key steps in nuclear transfer cloning. Although microtubule interrupters have been successfully used for oocyte enucleation, their potential side effect on oocyte developmental potential should be considered, and less harmful drugs should be explored for chemical-assisted enucleation. Based on our previous findings that any maturation promoting factor-activating agent induces ooplasmic protrusion without disrupting microtubules, we have studied the feasibility to use caffeine or MG132 for chemical-assisted enucleation. Experiments using goat oocytes showed that treatments for 30 min with 1-mM caffeine or 5-μM MG132-induced ooplasmic protrusions in about 85% of the oocytes, a percentage similar to that achieved with optimal demecolcine treatment. Rates of enucleation, cell fusion and in vitro blastulation were similar among caffeine, MG132, and demecolcine enucleation but significantly higher than blind aspiration. Furthermore, neither rates of pregnancy on days 90 and 120 nor the general rate of live births/embryos transferred differed significantly (p > 0.05) between caffeine and demecolcine enucleation. Although oocytes treated with caffeine did not retract protrusions until 2 h, many oocytes treated with MG132 withdrew protrusions as early as 0.5 h after treatment. The optimal treatment to induce ooplasmic protrusion in 75% pig oocytes was 8-mM caffeine for 60 min. Mouse oocytes responded poorly to demecolcine or caffeine with less than 40% forming inconspicuous protrusions following optimal treatments. It is concluded that caffeine can be used for enucleation of goat and pig oocytes with similar results as demecolcine, and live kids were born after caffeine-assisted enucleation.
Cellular reprogramming. 03/2011; 13(3):225-32.
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ABSTRACT: Dairy goats are ideal for the transgenic production of therapeutic recombinant proteins. The use of recombinant somatic cell lines for nuclear transfer (NT) allows the introduction of genes by transfection, increases the efficiency of transgenic animal production to 100%, and overcomes the problem of founder mosaicism. Although viable animals have been cloned via NT from somatic cells of 11 species, the efficiency has been extremely low. Both blastomere and somatic cell NT increased fetal loss and perinatal morbidity/mortality in cattle and sheep, but fetal loss and perinatal mortality appear to be relatively low in goats. In this study, we produced cloned goats by NT from cumulus cells and long-term cultured fetal fibroblast cells (FFCs) to abattoir-derived oocytes. NT embryos were constructed from electrofusion of cumulus cells (CCs), FFCs, or skin fibroblast cells (SFCs) with cytoplasts prepared from abattoir-derived ovaries. The NT embryos were activated with an optimized activating protocol (1 min exposure to 2.5 microM ionomycin followed by 2 hr incubation in 2mM 6-DMAP). Two viable cloned kids from CCs and one from long-term cultured FFCs (at passage 20-25) were born. Microsatellite analysis of 10 markers confirmed that all cloned offspring were derived from corresponding donor cells. To our knowledge, the production of cloned goat offspring using abattoir-derived oocytes receiving nuclei from CCs and long-term cultured FFCs has not been reported. The production of viable cloned animals after activation with reduced intensity of ionomycin and 6-DMAP treatment has also not been reported. Loss of cloned embryos was obvious after 45 and 90 days of pregnancy, and a lack of cotyledons, heart defects, and improperly closed abdominal wall were observed in the aborted fetuses and one cloned kid. The fusibility and in vitro developmental potential of embryos reconstructed from FFCs at passage 20-25 were significantly lower than those of embryos reconstructed from FFCs at passage 3-5, and the cloning efficiency of the long-term cultured cells was low (0.5%).
Molecular Reproduction and Development 08/2006; 73(7):834-40. · 2.53 Impact Factor
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ABSTRACT: Although ethylene glycol (EG) has been widely used for embryo cryopreservation in domestic animals, few attempts were made to use this molecule to freeze mouse and human embryos. In the few studies that used EG for slow-freezing of mouse and human embryos, complicated protocols for human embryos were used, and the protocols need to be simplified. Besides, freezing mouse morula with EG as a cryoprotectant has not been reported. In this paper, we studied the effects of embryo stages, EG concentration, duration and procedure of equilibration, sucrose supplementation and EG removal after thawing on the development of thawed mouse embryos, using the simple freezing and thawing procedures for bovine embryos. The blastulation and hatching rates (81.92% +/- 2.24% and 68.56% +/- 2.43%, respectively) of the thawed late compact morulae were significantly (P < 0.05) higher than those of embryos frozen-thawed at other stages. When mouse late compact morulae were frozen with different concentrations of EG, the highest rates of blastocyst formation and hatching were obtained with 1.8mol/L EG. The blastulation rate was significantly higher when late morulae were equilibrated in 1.8 mol/L EG for 10 min prior to freezing than when they were equilibrated for 30 min, and the hatching rate of embryos exposed to EG for 10 min was significantly higher than that of embryos exposed for 20 and 30 min. Both rates of blastocyst formation and hatching obtained with two-step equilibration were higher (P < 0.05) than with one-step equilibration in 1.8 mol/L EG. Addition of sucrose to the EG-based solution had no beneficial effects. On the contrary, an increased sucrose level (0.4 mol/L) in the solution impaired the development of the frozen-thawed embryos. In contrast, addition of 0.1 mol/L sucrose to the propylene glycol (PG)-based solution significantly improved the development of the frozen-thawed embryos. Elimination of the cryoprotectant after thawing did not improve the development of the thawed embryos. The cell numbers were less (P < 0.05) in blastocysts developed from the thawed morulae than in the in vivo derived ones. In summary, embryo stage, EG concentration, duration and procedure of equilibration and sucrose supplementation had marked effects on development of the thawed mouse embryos, and a protocol for cryopreservation of mouse embryos is recommended in which the late morulae are frozen in 1.8 mol/L EG using the simple freezing and thawing procedures of bovine embryos after a two-step equilibration and the embryos can be cultured or transferred without EG removal after thawing.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 09/2005; 21(5):766-72.
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ABSTRACT: ABSTRACT Effects of sperm and oocyte quality control on the efficiency of ICSI of in vitro matured goat oocytes were studied in this paper. The results showed that when injected intracytoplasmically, spermatozoa from caput, corpus and cauda epididymidis resulted in similar rates of fertilization, cleavage and morulae/blastocysts, but when injected subzonally, spermatozoa from caput and corpus gave rise to significantly lower rates of fertilization and embryo development than spermatozoa from the cauda epididymidis and ejaculates. When dead spermatozoa collected from semen that had been preserved in different ways were used for ICSI, those dead from liquid storage at 20 degrees C for 24 h gave rise to the best, but those dead from liquid storage at 5 degrees C for 15 days produced the poorest fertilization and embryo development. When spermatozoa were treated with different concentrations of Triton X-100 before ICSI, significantly higher rates of fertilization, cleavage and morulae/blastocysts were obtained with 0.0005% Triton X-100 than with other concentrations and manual immobilization. Oocytes were classified as of good and poor qualities by treatment in hypertonic sucrose solution, and rates of fertilization and embryo development were significantly higher in the good than in the poor oocytes after ICSI. Post-injection activation of oocytes with either A23187 or ionomycin/6-DMAP significantly increased the rates of fertilization, cleavage and morulae/blastocysts after ICSI. It is therefore concluded that (i) epididymal maturation mainly endowed spermatozoa with the capacity to fuse with the egg plasma membrane; (ii) different methods of semen storage caused different impairment of sperm fertilizing capacity; (iii) pre-injection treatment of spermatozoa with proper concentrations of Triton X-100 might be used to replace manual immobilization for ICSI; (iv) oocyte quality was a major factor influencing the efficiency of ICSI; (v) post-injection activation treatment of oocytes improved fertilization and embryo development after ICSI.
Shi yan sheng wu xue bao 11/2004; 37(5):367-74.
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ABSTRACT: To study the effect of post-treatment with 6-Dimethylaminopurine (6-DMAP) on oocyte activation and development, mouse oocytes collected at different times post human chorion gonadotropin (hCG) injection were incubated in 6-DMAP-containing Chatot-Ziomek-Bavister (CZB) medium for different periods after ethanol exposure, and activation and development were observed. When oocytes were cultured in 6-DMAP without prior ethanol exposure, the highest activation rate was only 40%. Incubation in 6-DMAP for 6 h following ethanol exposure significantly (P < 0.05) increased the activation rate in oocytes recovered 15 and 18 h post hCG, but this effect was not significant in the 21 h oocytes. When oocytes were incubated in 6-DMAP for 1 h at different time points after ethanol, a 6-DMAP susceptible temporal window was found to be located from the second to the fifth h in the 18 h oocytes and from the fourth to the fifth h in the 15 h oocytes, and within the window, the duration of 6-DMAP incubation can be reduced to 0.5 h with more than 80% activation. With the 13 h oocytes, however, 6-DMAP-incubation can only be shortened to 3 h and no specific temporal window was identified. Oocytes that were incubated in 6-DMAP for 1 or 2 h after ethanol exposure developed to morula/blastocyst stages at significantly (P < 0.05) higher rates than those incubated in 6-DMAP for 6 h. Our results suggested that (i) long duration of 6-DMAP incubation impaired the development of mouse parthenogenotes; (ii) the effect of 6-DMAP alone was limited without prior ethanol exposure; (iii) the egg age affected both the timing of 6-DMAP susceptibility and the duration of exposure required to obtain a maximal activating effect; (iv) the most effective activating protocols varied for oocytes of different ages.
Journal of Experimental Zoology Part A Comparative Experimental Biology 10/2004; 301(10):837-43.
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ABSTRACT: Systematical studies are lacking on the influencing factors and mechanisms of the heparin enhanced sperm capacitation, although many studies have shown that heparin enhanced sperm capacitation. The effect of heparin concentration and exposure time, incubation temperature and co-culture with oviductal epithelial cells or cumulus cells on goat sperm capacitation were investigated in this study. The motility, membrane and acrosome integrity and capacitated percentage of goat spermatozoa were assessed after different heparin treatments, and rates of fertilization and embryo cleavage were compared after in vitro insemination of oocytes with spermatozoa capacitated by different heparin treatments. The major results are summarized as follows: 1) When spermatozoa were capacitated with heparin at 5, 10, 25, 50 and 100 microg/mL for 45 min, 50 and 100 microg/mL heparin treatments produced the highest capacitated percentages of 55% and 56%, respectively, but the percentage of spermatozoa with intact acrosomes in the 100 microg/mL heparin treatment decreased significantly (P < 0.05) in comparison with that in the control group, indicating that the optimal heparin concentration for goat sperm capacitation would be 50 microg/mL. 2) Capacitated percentage of spermatozoa increased with extension of treatment time when goat sperm were treated with 50 microg/mL heparin for 0, 10, 20, 30, 45, 60 or 120 min. Although heparin treatments for 45 to 120 min did not differ significantly (P > 0.05) in capacitated sperm percentages, sperm motility and membrane integrity decreased significantly when treated with heparin for 120 min. This suggested that the optimal exposure time of heparin at 50 microg/mL for goat sperm capacitation would be 45 to 60 min. 3) Significantly higher capacitated percentages of spermatozoa were obtained when goat sperm were treated at 42 and 38.5 degrees C than at 15 and 37 degrees C, but sperm motility and acrosome integrity were significantly lower when spermatozoa were treated at 42 degrees C than they were treated at other temperatures. Temperature of 38.5 degrees C would, therefore, be the optimal temperature for goat sperm capacitation. 4) The capacitated percentage of spermatozoa was significantly higher when goat sperm were co-cultured with oviductal epithelial cells than when treated with heparin alone or co-cultured with cumulus cells, but sperm motility and membrane and acrosome integrity did not differ significantly among the three treatments. Rates of fertilization (91.3%) and cleavage (72.2%) were significantly higher in the oviductal epithelial cell co-culture group than those in the heparin alone group. This indicated that co-culture with oviductal epithelial cells significantly enhanced goat sperm capacitation by heparin treatment.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 04/2004; 20(2):252-6.
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ABSTRACT: The meiotic progression of goat oocytes from follicles of different diameters was investigated in this study. The results were summarized as follows: (1) The in vitro meiotic maturation capacity was different among oocytes from follicles of different diameters. And thus oocytes from < or = 0.5 mm follicles were unable to resume meiosis; oocytes from 0.8-1.2 mm follicles were capable to resume meiosis, but could develop only to MI stage (60% at 24 h); oocytes from 1.5-5 mm follicles had acquired full-meiotic maturation capacity and 91% of them developed to M II stage at 24 h of culture. (2) The percentage of oocytes with intact-germinal vesicles from 1.5-5 mm follicles decreased significantly during 2-8 h of in vitro maturation and the decrease was even more rapid during 4-6 h of culture (from 60% to 19%, p < 0.0005). The percentage of oocytes at M I-stage increased from 24% to 61% during 6-12 h of in vitro maturation, and it then decreased. By 24 h of culture, only 2% oocytes remained at M I-stage. Twenty one percent of the oocytes in this group developed to M II-stage at 16 h of culture, and by 24 h of culture, 91% were at M II-stage. (3) Statistic analysis of the meiotic progression (the duration of each cell cycle stage) of oocytes from 1.5-5 mm follicles showed that GV stage lasted from 0 to 3 h of culture, prometaphase-I stage was from 3.0 to 7.0 h, metaphase-I stage was from 7.0 to 14.6 h, anaphase-I/telophase-I was from 14.6 to 18.4 h and metaphase-II stage lasted from 18.4 to 24 h. (4) Whether the oocytes capable of GVBD and entrance of M I developed to M II, the timing of meiotic progression prior to M I was similar. In summary, our results provided necessary data for studies on the mechanisms and control of meiosis in mammalian oocytes.
Shi yan sheng wu xue bao 03/2004; 37(1):9-14.
