Comparative aspects of somatic cell nuclear transfer with conventional and zona-free method in cattle, horse, pig and sheep

Nuclear transfer (NT) is a complex procedure that requires considerable technical skills. Over the years attempts have been made to simplify the micromanipulations involved and to make the procedure more user-friendly. A significant step forwards has been the development of the zona-free NT methods. We have used zona-free NT with mechanical aspiration of the metaphase plate as a mean of enucleation, in a comparative approach with the conventional nuclear transfer zona-enclosed method in cattle, horse, sheep and pig. The absence of the zona considerably facilitates the enucleation step and significantly increases cell fusion success. On the other hand, the culture of zona-free NT embryos requires the embryos to be cultured individually or anyway separated from each other to avoid aggregation and also requires to prolong the in vitro culture up to the blastocyst stage before transfer. Blastocyst rate is equal or higher with zona-free method as compared to zona-enclosed method while survival after cryopreservation and development to term is comparable. In conclusion, our findings, together with published data, demonstrate that the zona-free system described in this paper can significantly increase the output of NT blastocysts over the conventional zona-enclosed system.     

Catalytic properties of the membrane enzyme prostaglandin H synthetase in a system of inverted micelles of aerosol OT in octane

Prostaglandin H synthetase solubilized in octane with the aid of hydrated reversed micelles of Aerosol OT (AOT) exhibits a catalytic activity dependent on hydration of the surface-active substance and its concentration. The maximum rate of the reaction is attained at the H2O/AOT molar ratio equal to 20 and amounts to a value close to the one observed in an aqueous solution. The inactivation rate of the enzyme in the course of the reaction does not depend on the water content in the system and is described by a Kin value commensurable with the Kin in an aqueous solution.     

Trypsin-like proteinase from Actinomyces fradiae 119

Using electrophoresis and ultracentrifugation, a homogeneous proteinase was isolated from protofradine, a protease Act. fradiae 119 preparation. The purification procedure included filtration on DEAE-cellulose, gel filtration through Arcylex P-10, CM-chromatography and desalting on Sephadex G-15. The proteinase under study is an endopeptidase which hydrolyzes low molecular weight synthetic trypsin substrates as well as casein and denaturated collagen. Diisopropylfluorophosphate and soya bean trypsin inhibitor completely inhibit the proteinase activity, whereas pCMB and EDTA have no such effect. The stability maximum is observed at pH of 2.5-3.5, the action maximum at pH 8.7-9.5. The amino acid composition of the enzyme is similar to that of trypsin from Str. griseus. The molecular weights of the proteinase as determined by gel filtration and sedimentation equilibrium method are equal to 25400 and 26500, respectively. The isoelectric point lies at 5.3. The data obtained suggest that the proteinase can be attributed to the family of trypsin proteinases.     

Regulation of c-Fos expression in the rat olfactory bulbs during olfactory learning

The distribution of c-Fos-immunopositive neurons was examined in the mitral/tufted and granular cell layers in the medium part of the main olfactory bulbs of 18-day-old rats after they had been trained for propionic acid vapour-guided search for dam in the Y-maze. On the next day these pups exhibited a strong preference for the propionic acid odor as compared to the control pups trained for this task without the odor cue and odor-familiarized pups exposed to propionic acid as a novel neutral stimulus. Exposure to propionic acid produced a moderate activation of c-Fos expression, mainly in the granular layer of the dorsomedial part of the bulb. Training in the Y-maze devoid of odor cues resulted in diffuse increase in the number of c-Fos-positive neurons both in the mitral and granular cell layers in all parts of the olfactory bulb. Maze training with the odor cue produced activation of c-Fos expression (which significantly exceeded the non-odor Y-maze group) in the dorsomedial olfactory bulb. These data suggest that associative olfactory conditioning results in activation of c-Fos expression that combines the effect of diffuse motivational excitation and specific olfactory input to the neurons which process odor cues.     

The effect of serum starvation on the efficacy of the nuclear reprogramming of rabbit embryonic fibroblasts

Successful transplantation of mammalian nuclei from differentiated cells has become possible after the application of original methods directed at the synchronization of cell cycles of the donor cell and recipient cytoplasm. We obtained a line of rabbit fetal fibroblasts which was used to study factors affecting the success of reprogramming. The nuclei of fetal fibroblasts (up to the 10th passage inclusive) proved to be capable of reprogramming and ensuring development of the cloned embryos until the preimplantation stages. The influence of synchronization of the cell cycles of the nucleus donor and recipient on the efficiency of reprogramming was studied. The rate of development of the cloned rabbit embryos to the morula-blastocyst stage reached 67% when the nuclei used were from stationary culture cells (G0-phase).     

Effect of melafen on the function of endoplasmic reticulum and plasmalemmal H+ -ATPase in the regulation of growth processes in potato tubers

The mechanism of the stimulatory effect of melafen on potato tuber sprouting was studied. The treatment with 10(-8) M melafen intensified division and stretching and activated granular endoplasmic reticulum of apical meristem cells. An increase in the activity of membrane-bound H+-ATPase in the plasmalemma of parenchymal cells of melafen-treated potato tubers and enhancement of passive proton permeability of the plasmalemma was observed. In vitro studies showed that melafen at concentrations of 10(-5-10-12) M stimulated the activity of plasmalemmal H+-ATPase in a concentration-dependent manner.