Biomedical subjects

Z Machaty

Publications and source records attributed to Z Machaty.

3 recordsLinked to original sources

PubMed2000

Production of transgenic porcine blastocysts by nuclear transfer.

In this study the in vitro development of porcine nuclear transfer (NT) embryos was investigated. Transgenic fetal fibroblast cells that were frozen after 5 days of serum starvation were injected immediately after thawing into enucleated metaphase II (MII) oocytes. Reconstructed embryos were activated by incubation in 200 microM thimerosal followed by a 30-min treatment of 8 mM DTT. The embryos were subsequently cultured in NCSU23, supplemented with 4 mg/ml BSA for 7 days. The actual cleavage rate (embryos showing > or =2 nuclei) in 6 replicates was 33% (ranging from 15% to 50%). Three blastocysts with cell numbers of 14, 15, and 18 were obtained. The blastocyst rate was significantly lower for NT embryos as opposed to parthenogenetically activated embryos (1% vs. 5%; P<0.05). The neomycin-resistance gene was amplified by PCR in all three NT embryos, indicating their origin from the injected transgenic fibroblasts. Efforts are now being directed in improvements in the nuclear transfer technology, whereby viable fetuses or offspring can be produced from these NT-embryos.

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PubMed1999

Activation of porcine oocytes with calcium ionophore: effects of extracellular calcium.

The present study examined the mechanism of A23187-induced activation in pig oocytes, with special reference to the effects of extracellular calcium on oocyte activation. The following endpoints were evaluated: intracellular free calcium concentration ([Ca2+]i), intracellular pH ([pH]i), cortical granule (CG) exocytosis, pronuclear formation, and blastocyst development. In experiment one, when oocytes were exposed to 50 microM A23187 for 5 min in a medium with, or without, calcium, a significant (P < 0.004) increase in the [Ca2+]i was observed in medium with calcium but not in medium without calcium. An increased [pH]i (0.08 unit in medium with calcium and 0.13 unit in medium without calcium), cortical granule exocytosis and pronuclear formation were observed in oocytes treated with A23187 irrespective of the presence or absence of calcium in the medium. In experiment two, the effects of treatment time (0, 0.5, 1, 2, and 5 min) on nuclear activation of oocytes with A23187 were further examined in medium with, or without, calcium. It was found that a 2 min treatment activated more (71-74%) oocytes than the other treatments. Treatment for 5 min in medium without calcium resulted in chromatin condensation in some oocytes. Microtubules were not found in these oocytes. In experiment three, developmental ability was examined of the oocytes treated with A23187 in medium with, or without, calcium. In vitro fertilized oocytes were used as a positive control. It was found that 16%, 6% and 38% of the oocytes treated with A23187 in medium with calcium, in medium without calcium, and in vitro fertilized oocytes developed to blastocysts after culture for 7 days, respectively. These results indicate that A23187 can induce pig oocyte activation in calcium-free medium without a typical increase in the [Ca2+]i and that A23187-induced pig oocyte activation is accompanied by an increase in [pH]i. Oocytes activated with A23187 can develop to blastocysts regardless of activation in medium with, or without, calcium.

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PubMed1996

gamma-Glutamyl transpeptidase of spermatozoa may decrease oocyte glutathione content at fertilization in pigs.

The presence of gamma-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'-O-(3'-thiotriphosphate) (GTP-gamma-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-gamma-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-gamma-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-gamma-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.

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