Volume 51 (2005) No. 2

Volume 51 (2005) No. 2
Original Articles
Effect of Protein Supplement Source on Porcine Oocyte Maturation and Subsequent Embryonic Development after Parthenogenetic Activation
V. KRYLOV1,2, R. KŘEN1, K. OKADA1, I. VACKOVÁ1, T. TLAPÁKOVÁ2, J. FULKA3.............................29
1Center for Cell Therapy and Tissue Repair, VUZV Labs., Prague, Czech Republic
2Charles University in Prague, Faculty of Science, Prague, Czech Republic
3Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic,  Liběchov, Czech Republic
Corresponding author: Vladimír Krylov, Charles University in Prague, Faculty of Science, Viničná 7, 128 43, Prague 2, Czech Republic, e-mail: vkrylov@natur.cuni.cz
Abstract.
Full text. 29-33
An Attempt to Reduce Polyspermic Penetration in Lamb Oocytes
T. SLAVÍK1, M. LIBIK2, E. WIERZCHOS2, J. FULKA1.................................................................34
1Institute of Animal Physiology and Genetics, Liběchov, Czech Republic
2University of Agriculture, Department of Sheep and Goat Breeding, Krakow, Poland
Corresponding author: Tomáš Slavík, Institute of Animal Physiology and Genetics, 277 21 Liběchov, Czech Republic. E-mail: slavik@iapg.cas.cz
Abstract.
Full text. 34-39
Morphometric Analysis of a Population of Diplopods of the Genus Rhinocricus Karsch, 1881
I. B. CALLIGARIS1, L. BOCCARDO2, M. R. SANCHES3, C. S. FONTANETTI1.................................40
1Departamento de Biologia, Universidade Estadual Paulista, Câmpus de Rio Claro, São Paulo, Brazil
2Departamento de Ciências Biológicas, Universidade Estadual do Sudoeste da Bahia, Câmpus de Jequié, Bahia, Brazil
3Instituto de Matemática e Estatística, Universidade de São Paulo, São Paulo, Brazil
Corresponding author: Izabela Braggião Calligaris, Instituto de Biociências, Departamento de Biologia, Universidade Estadual Paulista, Avenida 24-A no  1515 – CEP 13506-900, Câmpus de Rio Claro, São Paulo, Brazil. E-mail: icallig@rc.unesp.br
Abstract.
Full text. 40-46
Short Communications
The eNOS Gene Polymorphism Does Not Have a Major Impact on Lipid Parameters and Premature Coronary Artery Disease in Slovene Men (Caucasians)
A. MILUTINOVIĆ1, H. HRUŠKOVIČEVA1, 2...........................................................................47
1Institute of Histology and Embryology, Medical Faculty, University of Ljubljana,
2Division of Medical Genetics, Department of Obstetrics and Gynecology, Medical Centre Ljubljana, Slovenia
Corresponding author: Aleksandra Milutinović, Institute of Histology and Embryology, Medical Faculty of Ljubljana, Korytkova 2, 1105 Ljubljana, Slovenia. Tel.: (+386) 1 543 7382; Fax: (+386) 1 543 7361; e-mail: sandramilutinovic@yahoo.com
Abstract.
Full text. 47-49
Monoclonal Antibody Register
Specification of the Monoclonal Antibody PK1 Reactivity in Chinese Hamster Ovary Cells
J. HAŠEK1, L. PEŘINKA2, L. VALÁŠEK1..........................................................50
1Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
21st Faculty of Medicine, Charles University, Prague, Czech Republic
Corresponding author: Jiří Hašek, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague, Czech Republic. Fax: (+420) 241 062 501;  e-mail: hasek@biomed.cas.cz.
Abstract.
Full text. 50-51
Original Articles
Effect of Protein Supplement Source on Porcine Oocyte Maturation and Subsequent Embryonic Development after Parthenogenetic Activation
V. KRYLOV, R. KŘEN, K. OKADA, I. VACKOVÁ, T. TLAPÁKOVÁ, J. FULKA

The aim of this study was to compare the effect of purified GPBoS and commonly used FCS on porcine oocyte maturation and subsequent embryonic development after their parthenogenetic activation. COCs were obtained from dissected follicles and cultured for 18, 24, 30, 36, 42 and 48 h in M-199 medium either with GPBoS or FCS. After 24 h with GPBoS, 91% of oocytes reached MI stage while in the medium supplemented with FCS, only 29% of oocytes reached the same stage (P < 0.05). The majority of oocytes from the FCS group (61%) reached MI stage approximately 6 h later. In the time periods between 36 to 48 h both groups of oocytes reached the same stage of maturation. After 48 h of culture the oocytes with extruded polar bodies were activated by a single electric pulse and then cultured with 4 mM 6-DMAP. Activated oocytes were cultured in PZM-3 medium supplemented with 3 mg/ml of BSA. After 7 days, the development and the quality of embryos were evaluated. The results showed that the maturation of oocytes in the presence of GPBoS significantly increases their subsequent developmental ability when compared with FCS supplementation (27% vs. 19% of blastocysts, P < 0.05). However, differential staining revealed that once blastocysts are formed in either group, they had the same total cell number (40 vs. 41) and also the ICM/total cell ratio (0.27 vs. 0.29).
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An Attempt to Reduce Polyspermic Penetration in Lamb Oocytes
T. SLAVÍK, M. LIBIK, E. WIERZCHOS, J. FULKA

The incidence of polyspermy in lamb oocytes matured and fertilized in vitro is very high and this results in a reduced developmental potential of embryos arising from them. We have attempted to produce oocytes more resistant to this fertilization anomaly. The oocytes from prepubertal lambs 7–12 weeks old were matured in a medium supplemented with various blood sera and oviductal fluid and fertilized in vitro. Significantly higher monospermic penetration was found in a medium supplemented with BSA – 3 mg/ml (63.9%) and OF (20%) concentration (55.8%). Lower monospermy was recorded in the presence of 10% LS (44.6%) or 10% SS (40.8%), and particularly in a medium with 10% FCS (26.9%). In contrast, high monospermy (78.7%) was observed in oocytes from adult donors matured and fertilized in an identical system. In another set of experiments we estimated whether polyspermy can be reduced by improvement of the cytoplasmic maturation of prepubertal oocytes using a two-step maturation protocol.  After artificial arrest of the maturation for 24 h with a specific cdk inhibitor – BL-I, 50 μM – more than 80% oocytes from prepubertal and adult donors-did not resume meiosis. When incubated thereafter in a drug-free medium for another 24 h, the oocytes of both categories progressed to MII in the rate comparable with control (80% to 90% MII). However, after fertilization no significant differences in the level of monospermic penetration was recorded between the arrested group (59.8%) and control (58.8%), both matured in the presence BSA, and 46.6% and 52.3% after treatment with OF. Also, no significant difference was observed between the arrested and control oocytes from adult donors (72.6% and 84.8%, respectively). These results suggest that high polyspermy in prepubertal oocytes is caused by developmental imperfection and can’t be fully eliminated either by modifying the composition of culture media or by prolongation of the culture interval.
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Morphometric Analysis of a Population of Diplopods of the Genus Rhinocricus Karsch, 1881
I. B. CALLIGARIS, L. BOCCARDO, M. R. SANCHES, C. S. FONTANETTI

Diplopods belonging to the subclass Helminthomorpha may present one or both leg pairs of the seventh diplosegment modified into structures that aid copulation, called gonopods. These structures are used as a taxonomic trait for the description of most species. In the genus Rhinocricus  these structures are closely similar, so that it is difficult to distinguish species only on the basis of this trait. Two species, R. padbergi and R. varians, are found in the same habitat and present gonopods practically identical in shape; together they present a broad colour gradient, ranging from dark brown to light beige. Morphometric data for individuals of the experimental group were submitted to ANOVA (analysis of variance) and MANOVA, using Hotelling-Lawley Trace and generalized Mahalanobis distances (D2) tests. The results demonstrated a relationship between size and colour, with darker individuals being larger. On the basis of this preliminary analysis, we may suggest that the two species are distinct since dark individuals are distant from medium- and light-coloured individuals according to the D2 values. This seems to indicate a possible polymorphism of individuals belonging to R. padbergi which present close proximity in the values obtained. In all analyses, we observed that the main variables were diameter, length and telson size.
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Short Communications
The eNOS Gene Polymorphism Does Not Have a Major Impact on Lipid Parameters and Premature Coronary Artery Disease in Slovene Men (Caucasians)
A. MILUTINOVIĆ, H. HRUŠKOVIČEVA

eNOS affects the NO level in the blood vessel wall, and therefore eNOS might be considered as a candidate gene for CAD. In this cross-sectional case-control association study we tested the hypothesis whether the eNOS 4a/b gene polymorphism is a genetic marker for premature CAD in Slovene men. The eNOS 4a/b gene polymorphism was tested in 403 Slovene men: 215 cases with premature CAD and 188 subjects with no history of CAD. The frequency of 4a/b genotypes did not differ between patients and controls: in CAD patients the frequencies of the 4aa, 4ab, or 4bb genotype were 5.0%, 27.9%, or 67.1%, respectively, and in controls the genotype frequencies were 5.3%, 30.9%, or 63.8%, respectively. In this study the aa genotype of the eNOS 4a/b polymorphism was not associated with premature CAD (OR = 1, 95% CI 0.4–2.3, P = 0.9). Moreover, there were no differences in lipid parameters (total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol and triglycerides) between the subjects with the aa genotype and the subjects with the ab or bb genotype. In conclusion, we failed to demonstrate that the eNOS 4a/b gene polymorphism was a genetic marker for premature CAD in Slovene men.
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Monoclonal Antibody Register
Specification of the Monoclonal Antibody PK1 Reactivity in Chinese Hamster Ovary Cells
J. HAŠEK, L. PEŘINKA, L. VALÁŠEK

Background. eIF3a/Tif32/Rpg1 is the evolutionarily conserved subunit of yeast eukaryotic translation initiation factor 3 (eIF3) that promotes multiple steps of the initiation pathway resulting in formation of the 48S pre-initiation complex at the AUG start codon of mRNA.
The monoclonal antibody PK1 (IgG1 kappa) (Jiřincová et al., 1998) against the recombinant protein Rpg1 (Kovarik et al., 1998) has already proved itself as a useful tool for analysing the eIF3 complex in S. cerevisiae (Valášek et al., 1998). It binds either to native or denaturated epitopes of Rpg1p and it can be used to efficiently isolate the eIF3 complex by immunoprecipitation (Valášek et al., 1999). This antibody was also used to show by immunofluorescence that yeast eIF3a is a microtubule-interacting protein (Hašek et al., 2000). In order to test whether this characteristic of yeast eIF3a is also conserved in higher eukaryotes, we analyzed the immunoreactivity of the anti-Rpg1p monoclonal antibody (PK1) in Chinese hamster CHO, human HeLa and mouse 3T3 cells.
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