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The objective of this study was to determine the effects of oviductal fluid and heparin on sperm penetration and the characteristics of spermatozoa. The addition of oviductal fluid and heparin to the fertilisation medium decreased sperm penetration and the mean number of spermatozoa in penetrated eggs. The number of spermatozoa firmly bound to zona pellucida was also decreased in the presence of oviductal fluid and heparin. Chlortetracycline (CTC) fluorescence patterns were used to determine the incidence of capacitation and the acrosome reaction. The proportion of capacitated and acrosome-free spermatozoa increased when spermatozoa were exposed for 1.5 and 3 h to oviductal fluid and heparin. In contrast heparin alone did not increase the number of capacitated spermatozoa at these time points. These results suggest that factor(s) in oviductal secretions reduce polyspermic fertilisation and the number of spermatozoa that will penetrate porcine oocytes. The reduction of polyspermic penetration by oviductal secretions may be due to a reduced number of spermatozoa in the fertilisation medium with an intact acrosome.
The objective of this study was to determine developmental pattern and cell allocation to the inner cell mass and trophectoderm in haploid and diploid embryos following parthenogenetic activation. In vitro matured porcine oocytes were activated by ethanol treatment and cultured in the presence or absence of cytochalasin B for 5h. The oocytes were then cultured in the NCSU23 for 9 days. The combined treatment with cytochalasin B following ethanol treatment did not increase (p >0.1) the incidence of activation. The incidence of development to the blastocyst stage was higher (p <0.05) in the combined treatments of ethanol and cytochalasin B as compared with ethanol treatment alone. The percentage of oocytes with two female pronuclei was higher (p < 0.01) in oocytes treated with cytochalasin B than that in ethanol treatment alone. Treatment with both ethanol and cytochalasin B increased (p <0.01) the incidence of diploid chromosome spread over just the ethanol treatment alone. The average numbers of total cells and inner cell mass were significantly reduced (p <0.05) in the ethanol treatment alone as compared with the combined cytochalasin B and ethanol treatment. These results suggested that the ploidy may affect blastocoele formation and cell allocation to inner cell mass and trophectoderm in the pig.
In this study we imaged integral changes in microfilament assembly and cortical granule distribution, and examined effects of microfilament inhibitor on the cortical granule distribution during oocyte maturation, parthenogenetic activation and in vitro fertilisation in the pig. The microfilament assembly and cortical granule distribution were imaged with fluorescent-labelled lectin and rhodamine-labelled phalloidin under laser scanning confocal microscopy. At the germinal vesicle stage, cortical granule organelles were located around the cell cortex and were present as a relatively wide area on the oolemma. Microfilaments were also observed in a wide uniform area around the cell cortex. Following germinal vesicle breakdown, microfilaments concentrated in the condensed chromatin and cortical granules were observed in the cortex. Treatment with cytochalasin B inhibited microfilament polymerisation and prevented movement of cortical granules to the cortex. Cortical granule exudation following sperm penetration was evenly distributed in the entire perivitelline space. These results suggest that the microfilament assembly is involved in the distribution, movement and exocytosis of cortical granules during maturation and fertilisation.
Incomplete reprogramming of the donor cell nucleus after nuclear transfer (NT) probably leads to the abnormal expression of developmentally important genes. This may be responsible for the low efficiency of cloned animal production. Insulin-like growth factor 2 (IGF2) and IGF2 receptor (IGF2R) are imprinted genes that play important roles in preimplantation development. To obtain an insight into abnormal gene expression after nuclear transfer, we assessed the transcription patterns of IGF2-IGF2R in single in vitro fertilised and cloned embryos by reverse-transcription polymerase chain reaction (RT-PCR). IGF2R expression did not differ significantly but IGF2 was more highly expressed in cloned embryos than in IVF embryos (p < 0.05). This was confirmed by a quantitative RT-PCR method. Thus, incomplete reprogramming may induce abnormal transcription of IGF2 in cloned embryos.
In this study we examined the developmental potential of reconstructed embryos and the fate of donor mitochondria during preimplantation development after nuclear transfer in cattle. Isolated cumulus cells were used as donor cells in nuclear transfer. Cumulus cells labelled with MitoTracker Green FM fluorochrome were injected into enucleated bovine MII oocytes and cultured in vitro. MitoTracker labelling on donor cells did not have a detrimental effect on blastocyst formation following nuclear transfer. Cleavage rate was about 69% (56/81) and blastocyst formation rate was 6.2% (5/81) at 7 days after nuclear transfer. The labelled mitochondria dispersed to the cytoplasm and became distributed between blastomeres and could be identified up to the 8- to 15-cell stage. Small patches of mitochondria were detected in some 8- to 15-cell stage embryos (5/20). However, donor mitochondria were not detected in embryos at the 16-cell stage and subsequent developmental stages. In the control group, mitochondria could be identified in arrested 1-cell embryos up to 7 days after nuclear transfer. These results suggest that disappearance of the labelled donor mitochondria in nuclear transfer bovine embryos is not due to fading of the fluorochrome marker, but is rather an as yet undefined cytoplasmic event.
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