Production of ZFN-mediated GGTA1 knock-out pigs by microinjection of gene constructs into pronuclei of zygotes.

Animals as a source of organs and tissues for xenotransplantation could become a backup solution for the growing shortage of human donors. The presence of human xenoreactive anti- bodies directed against Galα1,3Gal antigens on the cell surface of a pig donor triggers the activa- tion of the complement leading to a hyperacute reaction. The development of genetic engineer- ing techniques has enabled the modification of genomes by knocking in and/or knocking out genes. In this paper, we report the generation of modified pigs with ZFN mediated disruption of the GGTA1 gene encoding the enzyme responsible for synthesis of Galα1,3Gal antigens. ZFN plasmids designed to target the exon 9 region of the pig GGTA1 gene encoding the catalytic domain were injected into the pronuclei of fertilized egg cells. Among 107 piglets of the F0 gene- ration analyzed, one female with 9-nt deletion in exon 9 of the GGTA1 gene was found. 13 of 33 piglets of the F1 generation represented the +/- GGTA1 genotype and 2 of 13 F2 piglets repre- sented the -/- GGTA1 genotype. No changes in the animals' behavior, phenotype or karyotype were observed. Analysis confirmed heredity of the trait in all animals. A complex functional analysis of the modified animals, including flow cytometry, human serum cytotoxicity test and immunohistochemical detection, was performed to estimate the phenotype effect of genetic modification and this indicated an efficient GGTA1 knock-out in modified pigs.

Identification of perivascular and stromal mesenchymal stem/progenitor cells in porcine endometrium.

Mammalian uterus contains a population of mesenchymal stem/progenitor cells that likely contribute to endometrial regeneration during each reproductive cycle. In human and mouse, they reside in perivascular, epithelial and stromal compartments of the endometrial functionalis and basalis. Here, we aimed to identify tissue resident cells expressing mesenchymal stem cell markers CD29, CD44, CD90, CD105, CD140b and CD146 in the porcine endometrium. We used single immunofluorescence and Western blotting. Each of these markers was detected in small cells surrounding endometrial blood vessels. CD105 and CD146 were also expressed in single stromal cells. A few stromal and perivascular cells showed the presence of pluripotency marker Oct4 in the cytoplasm, but not in the nucleus, which may imply they are not truly pluripotent. Endometrial cell cultures were examined for the expression of CD29, CD44, CD90, CD105 and CD140b proteins and tested in wound-healing assay and culture model of chemotaxis. In conclusion, our results demonstrate perivascular location of prospective mesenchymal stem/progenitor cells in the porcine endometrium and may suggest that stromal CD105+ and CD146+ cells represent more mature precursors originating from their perivascular ancestors.

Caspase-3 Expression and ALT, AST, and GGT Activity After 24 Hours of Porcine Liver Cold Storage, Depending on the Type of Transgenesis.

BACKGROUND: Because of an insufficient number of human organs for transplantation, xenotransplantation may become an effective alternative. We aimed to analyze if the type of transgenesis has an influence on the hepatic caspase-3 expression, the enzyme that executes apoptosis as well as ALT, AST, and GGT activity after 24 hours of cold storage. METHODS: The experiment was carried out on the 24 livers of Polish White Landrace pigs carrying human α1,2-fucosyltransferase and/or α-galactosidase (GAL) genes and livers without this genetic modification (control). Livers were perfused, stored for 24 hours in solution, and subsequently re-flushed. Hepatic concentration of the caspase-3 protein and its mRNA expression were measured just after the animal was killed as well as after 30 minutes of perfusion and after 24 hours of cold storage followed by 30 minutes of reperfusion. Caspase-3 mRNA level was detected with the RT-PCR method. Protein concentration (capsase-3 active and inactive) was assessed with the Western blotting technique. Kinetic methods were applied for the analysis of the ALT, AST, and GGT activity. RESULTS: The highest increase of the ALT activity after cold storage was observed in the group with GAL transgenesis, whereas the GGT activity was highest in the unmodified livers. There was no difference in the caspase-3 expression and AST activity after cold storage as compared with the respective initial results (P = .57 and P = .97, respectively). CONCLUSIONS: It appears that transgenesis does not aggravate ischemic injury of the liver.

Concentration of Tumor Necrosis Factor-α and Interleukin-1ß in Isolated Porcine Liver Depending on Type of Transgenesis.

BACKGROUND: Transgenic animals may serve as organ donors in human organ transplantation. However, the number of the studies addressing all doubts related to this issue is currently insufficient for the clinical application of this approach. The aim of this study was to analyze the hepatic tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) synthesis during a 24-hour cold preservation of the transgenic pig liver, depending on the type of transgenesis. MATERIALS AND METHODS: The study was carried out on wild-type and transgenic pig livers with transferred human α1,2-fucosyltransferase (FUT) and/or α-galactosidase (GAL) gene (four groups; n = 6). Harvested livers were perfused for 30 minutes and stored for 24 hours in Biolasol (Biochefa) solution at 4°C with a subsequent 30-minute reperfusion (reflush). TNF-α and IL-1ß concentrations were analyzed with an enzyme-linked immunosorbent assay. Perfusates were collected during the initial perfusion as well as after 24 hours of preservation and during the reperfusion. Tissue samples were harvested just after animal sacrifice, and after organ perfusion and reperfusion. RESULTS: A decrease in TNF-α concentration in homogenates was noted after both perfusion and reperfusion in all experimental groups. In contrast, there was a significant decrease in IL-1ß concentration in the group with combined human FUT and GAL transgenes. Concurrently, increases in TNF-α and IL-1ß concentrations were observed in the reperfusion perfusates in all groups. CONCLUSION: This study shows that IL-1ß is synthesized in the ischemic livers of the transgenic animals with both human α1,2-fucosyltransferase and α-galactosidase transgenes. Further analysis is required to determine the importance of this observation.

Biolasol: novel perfusion and preservation solution for kidneys.

INTRODUCTION: Biolasol solution (Pharmaceutical Research and Production Plant "Biochefa," Sosnowiec, Poland) is a novel extracellular perfusion and ex vivo hypothermic kidney preservation solution. It ensures maintenance of homeostasis, reduces tissue edema, has low viscosity, and allows the graft to preserve structural and functional integrity. It minimizes ischemia-reperfusion damage. METHODS: Perfundates from control and transplanted kidneys flushed with Biolasol or ViaSpan solutions (Arkas, Warszawa, Poland) were analyzed. Parameters of serum and urine collected from 12 pigs after auto-transplantation were also analyzed. Renal medulla was investigated for structural alterations by analyzing hematoxylin and eosin-stained slides. The mean survival time of pigs after the auto-transplantation procedure was the measure for the novel Biolasol solution effectiveness. RESULTS: We observed a statistically significant decrease in marker enzyme levels alanine transaminase, aspartate transaminase, lactic dehydrogenase, and ions (Na and K) in pigs with grafts flushed with Biolasol. Histopathologic examination revealed that the renal cortex structure was not damaged after the use of Biolasol solution. CONCLUSION: Biolasol solution protects kidneys against ischemia damage and does not differ significantly from the "golden standard" ViaSpan solution.

Cytochrome P450 3 A expression in pigs livers after 24-hour preservation in Biolasol solution depending on the type of transgenesis.

BACKGROUND: An insufficient number of organs for transplantation shows the need for the development of new technologies. Xenotransplantation might be the answer. OBJECTIVE: To determine if the type of transgenesis influences the level of CYP3A4, which takes an active part in xenobiotics metabolism in livers after 24-hour storage, depending on the kind of solution used for preservation. MATERIALS AND METHODS: The experiment was carried out on 30 livers of Polish White Landrace divided into 5 groups depending on transgene type. The following human genes were transferred: α1,2-fucosyltransferase (groups I and II), α-galactosidase (III), combined α1,2-fucosyltransferase/α-galactosidase transgene (IV), and livers without modification (V). The livers were perfused and subsequently stored for 24 hours in Ringer's solution (group I) or Biolasol solution (II-V). Reperfusion/reflush was performed. CYP3A29 isomer concentration was analyzed in liver specimens collected twice: 30 minutes after perfusion and 30 minutes after reperfusion/reflush. Expression of mRNA CYP3A29 was marked using RT-PCR analysis and of protein CYP3A29 using Western blotting technique. RESULTS: The most significant decrease in protein CYP3A29 expression after 24-hour preservation was observed in group I (55.88% decrease), while the least significant was observed in group IV (10.44% decrease). mRNA expression evaluation was similar: the most significant decrease was observed in group I (87.8% decrease) and the least significant in group III (4.6% decrease). CONCLUSION: α1,2-Fcosyltransferase transgene seems to influence mRNA and protein CYP3A expression in case of liver grafting and preservation for transplantation. CYP3A expression was also influenced by the kind of preservation solution used.

Interleukin-6 concentration in the transgenic pig's liver preserved for 24 hours in Biolasol solution.

INTRODUCTION: Increasing the human lifespan contributes to a higher number of patients with end-stage organ failure, which in turn stimulates the search for alternative sources. Xenotransplantation seems to be a promising approach in this respect. OBJECTIVE: Analysis of changes in interleukin (IL)-6 concentration during 24-hour preservation of transgenic swine livers, depending on the kind of transgenesis and preservation solution used. MATERIALS AND METHODS: The experiment was carried out in swine livers with transferred human genes that were divided into 5 groups. The following human genes were transferred: α1,2-fucosyltransferase (group I and II), α-galactosidase (III), combined α1,2-fucosyltransferase/α-galactosidase transgene (IV), and livers without modification (V). The livers were perfused and subsequently stored for 24 hours in Ringer's (group I) or Biolasol solutions (II-V). Reflush was then performed. IL-6 concentration was analyzed in the solution samples collected at the beginning and end of perfusion, and after 24 hours of preservation. ELISA was used to evaluate IL-6 concentration. RESULTS: In liver homogenates from group I, IL-6 concentration after 24 hours of preservation increased by 8.24% compared to the levels observed after perfusion, whereas in the other groups IL-6 concentration decreased. The most significant decrease, 49.51%, was observed in group II; the least significant in group IV, 10.72%. In case of supernatants, a statistically significant increase of AUC0-30min level in relation to perfusion was observed in every group after 24-hour preservation and reperfusion. The highest values of AUC0-30min were observed in group I (α1,2-fucosyltransferase, Ringer's solution). CONCLUSION: The study indicates the hepatoprotective action of Biolasol solution.

Transgenic pigs designed to express human α-galactosidase to avoid humoral xenograft rejection.

The use of animals as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human organ donors. However, the presence of xenoreactive antibodies in humans directed against swine Gal antigen present on the surface of xenograft donor cells leads to the complement activation and immediate xenograft rejection as a consequence of hyperacute reaction. To prevent hyperacute rejection, it is possible to change the swine genome by a human gene modifying the set of donor's cell surface proteins. The gene construct pGal-GFPBsd containing the human gene encoding α-galactosidase enzyme under the promoter of EF-1α elongation factor ensuring systemic expression was introduced by microinjection into a male pronucleus of the fertilised porcine oocyte. As a result, the founder male pig was obtained with the transgene mapping to chromosome 11p12. The polymerase chain reaction (PCR) analysis revealed and the Southern analysis confirmed transgene integration estimating the approximate number of transgene copies as 16. Flow cytometry analysis revealed a reduction in the level of epitope Gal on the cell surface of cells isolated from F0 and F1 transgenic animals. The complement-mediated cytotoxicity assay showed increased viability of the transgenic cells in comparison with the wild-type, which confirmed the protective influence of α-galactosidase expression.

Lipid content and cryotolerance of porcine embryos cultured with phenazine ethosulfate.

The addition of phenazine ethosulfate (PES) to culture medium was investigated for its effect on pig embryo development, apoptosis, cytoplasmic lipid content and survival after OPS vitrification. Porcine zygotes were cultured in NCSU-23 medium supplemented with 0 (control) or 0.05 microM PES up to the blastocyst stage and were vitrified using OPS technology. Culture of embryos with PES reduced the cytoplasmic lipid content, as measured by fluorescence of blastocysts stained with Nile Red. The survival rate of vitrified blastocysts was slightly enhanced, although not significantly, in the presence of PES compared to the PES-free group (45.2 and 37.9 percent, respectively). These results showed that culturing porcine embryos in medium with PES increased the proportion of morula and blastocyst formation and reduced the index of DNA fragmentation and the cytoplasmic lipid content of cultured blastocysts. However, the use of PES during in vitro culture had limited effect on porcine blastocyst survival after vitrification.

Apoptotic-like changes in the spermatozoa of fresh and stored boar semen and the quality of embryos produced in vivo.

The aim of this study was to determine the apoptotic-like changes in the spermatozoa of fresh and stored boar semen and to investigate the relationship between this phenomenon and the quality of embryos produced in vivo. The experiments were divided into two series. In the first series, ten ejaculates were collected from five boars, which were crossbreeds of the Polish Landrace and Large White breeds. The semen was stored as a liquid until Day A (the day on which sperm motility decreased to 30%). Three fluorescence methods were used to evaluate semen quality: an assay to assess the early changes in sperm membrane integrity using the fluorophore YO-PRO-1, an assay for phosphatidylserine (PS) translocation across the plasma membrane using fluorescein-labeled annexin-V and the mitochondrial-specific probe JC-1 (5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide) for measuring changes in mitochondrial membrane potential. Our results showed that liquid preservation of boar semen causes apoptotic-like changes in the sperm, and a significant increase in both: apoptotic sperm (YO-PRO-1(+)/PI(-)) and early apoptotic sperm (annexin-V(+)/PI(-)) were observed between Day 0 (fresh semen) and Day A only in semen from three of the five boars. In the second series of experiments, the semen from boar nos. 1, 2, and 3 was selected for insemination of superovulated gilts. The fertilizing capacity of fresh and stored semen with different levels of apoptotic spermatozoa was measured based on the morphology and the number of cells of embryos that were obtained after insemination with this semen. Our studies indicated no significant differences in the fertilization rate of gilts after insemination with fresh and stored semen with increased levels of apoptotic spermatozoa. After insemination with stored semen, a significantly greater number of degenerated embryos were observed, but the morphologically normal blastocysts obtained after insemination with either fresh or stored semen had a similar number of nuclei.

Mitochondrial activity and morphology in developing porcine oocytes and pre-implantation non-cultured and cultured embryos.

Mitochondria are important determinants of developmental competence for oocytes and embryos owing to their central role in cellular metabolism, yet mitochondrial activity and morphometry during early porcine development have not been quantified. In this study, we examined the membrane potential Δψ(m) and the surface density Sv(in,m) of the inner mitochondrial membrane in pig oocytes and pre-implantation embryos using fluorescent probes and confocal microscopy. Mitochondria and their cristae were also examined by transmission electron microscope. Δψ(m) was consistently low from immature oocytes up to morulae and increased significantly in the early blastocyst before decreasing at the expanded blastocyst stage. This stage-dependent pattern of Δψ(m) changes differs from that reported for other mammals. We also determined that Δψ(m) is lower in cultured when compared to non-cultured porcine early blastocysts. Sv(in,m) was higher in immature oocytes than mature oocytes and remained constant up to the 4- to 8-cell embryo stage. It increased significantly at morula and early blastocyst stages. No differences in Sv(in,m) were found between developmentally matched non-cultured and cultured embryos. These results indicate that the inner mitochondrial membrane potential and surface density change significantly during pre-implantation porcine development in relation to metabolic alterations of the embryo. It is possible that modification of Δψ(m) by manipulating culture conditions may improve the performance of embryos that develop in vitro.

Changes of lipid composition in non-cultured and cultured porcine embryos.

The objective of the present study was to evaluate the lipid composition of cultured and non-cultured pig embryos during cleavage using histochemical methods. The authors studied pig zygotes as well as 2-to 4-cell embryos, morulae, and blastocysts that were either non-cultured or cultured in NCSU-23 medium. To detect different types of lipids, the authors used the Churukian method with Oil red O, the Sudan black B method, the Cain method with Nile blue sulfate, and the modified osmium tetroxide-ethanol treatment. In the zygotic lipid droplets, diverse classes of unsaturated and saturated lipids were found, with particularly high levels of unsaturated hydrophobic lipids, mainly triglycerides and other esters, free fatty acids, and phospholipids. In the zygotic cytoplasm, the authors observed high levels of fatty acids and phospholipids. The total lipid content remained constant up to the morula stage, decreasing later at the blastocyst stage, but the overall amount of unsaturated lipids declined earlier, at the 2-to 4-cell stage. The amount of free fatty acids and phospholipids decreased during cleavage in both non-cultured and cultured porcine embryos. The main differences between the non-cultured and cultured embryos were the more pronounced reduction in the amount of unsaturated hydrophobic lipids in droplets and the cytoplasmic free fatty acids observed in the cultured morula and the lower content of phospholipids in the cytoplasm of the cultured 2-to 4-cell embryos relative to the non-cultured embryos. The decrease in the unsaturated lipid, free fatty acid, and phospholipid content during in vivo development and the differences in the amount of these types of lipids between developmentally matched cultured and non-cultured pig embryos correlate well with modifications of lipid and carbohydrate metabolism.

Effect of semen quality in transgenic boars on the developmental competence of preimplantation embryos.

The aim of this study was to compare the fertilising capacity of sperm from 6 transgenic (TG) and 6 non-transgenic (NTG) boars based on analyses of embryos resulting from insemination with sperm from these particular boars. Expanded blastocysts were collected from five groups of synchronised gilts (six gilts per group) inseminated by TG boars bearing a gene construct containing the human alpha1,2-fucosyltransferase gene and by NTG boars. The ejaculates used for insemination were analysed to detect apoptotic changes using two fluorescence methods: an assay to assess early changes in the membrane integrity of the sperm using the YO-PRO-1 fluorophore and an assay for phosphatidylserine (PS) translocation across the plasma membranes using fluorescein-labelled Annexin-V. Our results, using a combination of YO-PRO-1 and PI fluorophores, revealed no significant differences in the percentage of sperm subpopulations between non-transgenic and transgenic boars (P<0.01). Moreover, the second fluorescent probe also revealed no significant differences between the average values of live (Ann-V(-)/PI(-)), early apoptotic (Ann-V(+)/PI(-)), and late apoptotic/early necrotic sperm (Ann-V(+)/PI(+)) as calculated for TG and NTG boars. Only the percentage of necrotic sperm (Ann-V(-)/PI(+)) was significantly different (P<0.05) between transgenic and non-transgenic boars (3.4%+/-2.7; 7.2%+/-2.1, respectively). The quality of the preimplantation embryos at the blastocyst stage was determined by counting the number of cells, observing a TUNEL-positive reaction and by caspase-3 labelling. We found that expanded blastocysts that were derived from gilts inseminated with TG and NTG boar semen showed almost no DNA fragmentation (80%) and 70% caspase-3 activity. The expanded blastocysts that were derived from gilts inseminated with TG and NTG boar semen did not differ significantly in their DNA fragmentation, and there were no differences in caspase-3 activity. These results revealed a positive correlation between the percentage of blastocysts with TUNEL-positive nuclei and the percentage of blastocysts with caspase-3 activity (r=0.9787; P<0.0001).

Assessment of plasma membrane and chromatin structure of sperm from transgenic and non-transgenic boars.

The aim of this study was to determine the apoptotic changes and chromatin damage in non-transgenic and transgenic boars carrying the human alpha1,2-fucosyltransferase gene. Five ejaculates were collected from six transgenic (TG) and six non-transgenic (NTG) boars. Five ejaculates were collected from six transgenic (TG) and six non-transgenic (NTG) boars both crossbreds of Polish Landrace and Large White. Two fluorescence methods were employed to measure apoptosis: an assay to assess the early changes in sperm membrane integrity using fluorophore YO-PRO-1 and an assay for phosphatidylserine (PS) translocation across the plasma membrane using fluorescein-labeled Annexin-V. The chromatin damage was assessed based on the sperm chromatin structure assay method. No significant differences in the proportion of all detected subpopulations of spermatozoa were found between TG and NTG boars. Similarly, the analysis of the chromatin structure revealed no statistical differences in the sperm chromatin damage between TG and NTG boars. In conclusion, the presence of the human alpha1,2-fucosyltransferase gene in the genome of TG boars did not cause any spermatogenesis process disturbances leading to the increased production of apoptotic spermatozoa. Moreover, the low level of sperm with damaged chromatin in TG boars confirms the high stability of the spermatogenesis process in the TG boars analyzed.

Lipid content of non-cultured and cultured pig embryo.

The objectives of the study were: (i) to work out a precise and efficient method for quantitative analysis of lipid content and (ii) to quantitatively determine the lipid content in non-cultured and cultured pig embryos. The experiment was carried out on pig embryos from zygote to late blastocyst stages produced in vivo and embryos collected at the zygote stage and then cultured in vitro up to blastocyst stage. Embryos were fixed, dehydrated, embedded in epoxy resin and cut into semi-thin sections to analyse the quantity of lipids in fat droplets. Stained sections were then analysed with Cavalieri and point counting methods to evaluate the following stereological parameters of the embryo: total embryo volume - V(e), volume density of cytoplasm per unit volume of embryo - Vv(c,e), volume density of lipid droplets per unit volume of embryo cytoplasm - Vv(fat,c) and total volume of lipid droplets per whole embryo - V(fat). Values of Vv(fat,c) and V(fat) remained unchanged up to the morula stage, but decreased significantly at blastocyst and late blastocyst stages both in cultured and non-cultured embryos. Volume density of lipid droplets per unit volume of embryo cytoplasm and total volume of lipid droplets for cultured embryos showed statistically significant differences between late blastocyst and almost all other stages. Comparisons of Vv(fat,c) in embryos at the same stages of development but differing in origin of embryos (non-cultured or cultured) show that statistically significant differences exist for all analysed stages. In conclusion, differences in lipid content observed in pig embryos were dependent on the developmental stage of the embryo as well as the culture conditions (i.e. cultured and non-cultured embryos at the same stage of development).

Generation of transgenic rabbits by the novel technique of chimeric somatic cell cloning.

A novel technique of chimeric somatic cell cloning was applied to produce a transgenic rabbit (NT20). Karyoplasts of transgenic adult skin fibroblasts with Tg(Wap-GH1) gene construct as a marker were microsurgically transferred into one, previously enucleated, blastomere of 2-cell non-transgenic embryos, while the second one remained intact. The reconstructed embryos either were cultured in vitro up to the blastocyst stage (Experiment I) or were transferred into recipient-females immediately after the cloning procedure (Experiment II). In Experiment I, 25/102 (24.5%) embryos formed blastocysts from whole embryos and 46/102 (44.12%) embryos developed to the blastocyst stage from single non-operated blastomeres, while the reconstructed blastomeres were damaged and degenerated. Thirteen (12.7%) embryos did not exceed 3- to 4-cell stages and 18 (17.7%) embryos were inhibited at the initial 2-cell stage. Out of 14 blastocysts which were subjected to molecular analysis, the transgene was detected in the cells of 4 blastocysts. In Experiment II, 163/217 (75.0%) embryos were transferred into 9 pseudopregnant recipient-rabbits (an average of 18 embryos per recipient). Four recipient-females (44.4%) became pregnant and delivered a total of 24 (14.7%) pups. Molecular analysis confirmed that two pups (1.2%), one live and one stillborn, showed a positive transgene signal. Live transgenic rabbit NT20 appeared healthy and anatomically as well as physiologically normal. The results of our experiments showed that transgenic adult skin fibroblast cell nuclei, which have been introduced into the cytoplasmic microenvironment of single enucleated blastomeres from 2-cell stage rabbit embryos, are able to direct the development of chimeric embryos not only to the blastocyst stage but also up to term.

Sequence analysis of proviral DNA of porcine endogenous retroviruses.

Among all species analyzed, the domestic pig seems to be the most appropriate organ donor for xenotransplantation. Porcine endogenous retroviruses (PERVs) are present in genomes of all pigs and are capable of infecting human cells in vitro thus posing a serious threat for xenotransplantation procedures. Despite the abundant distribution of PERVs integrated with porcine genome, the majority of PERV proviral DNA is not capable of expressing viral proteins unless seriously mutated. The aim of the study was to analyze PERV genome for mutations. The study was performed on blood samples from 146 pigs. Long-range polymerase chain reaction (Long-PCR) was performed with primer sets designed within long terminal repeats (LTRs). Long-PCR products of different molecular weights were obtained: 530 bp (33.1% of individuals), 580 bp (76.7%), 933 bp (100%), and 2900 bp (59.8%). Amplimers of 7200 bp were absent in 12.8% of individuals, indicating the lack of intact proviral DNA. Sequence analysis showed that most PERV proviral DNA was significantly mutated, thus suggesting the inability to express functional viral RNA; however, it cannot be ruled out that compensatory recombination processes could occur enabling replication of defective proviruses.

Flow cytometric cell cycle analysis of somatic cells primary cultures established for bovine cloning.

An important factor governing developmental rates of somatic cloned embryos is the phase of the cell cycle of donor nuclei. The aim of this experiment was to investigate the distribution of cell cycle phases in bovine cumulus and fibroblast cells cultured using routine treatment, and under cell cycle-arresting treatments. The highest percentages of cumulus cells in the G0 + G1 stage were observed in uncultured, frozen/thawed cells originating from immature oocytes (79.8 +/- 2.2%), fresh and frozen/thawed cells from in vitro matured oocytes (84.1 +/- 6.2 and 77.8 +/- 5.7%, respectively), and in cycling cells (72.7 +/- 16.3 and 78.4 +/- 11.2%, respectively for cumulus cells from immature and in vitro matured oocytes). Serum starvation of cumulus cultures markedly decreased percentages of cells in G0 + G1, and prolonged starvation significantly increased (P < 0.05) percentages of cells in G2 + M phase. Culture of cumulus cells to confluency did not increase percentages of cells in G0 + G1. Contrary to findings in cumulus cells, significantly higher percentages of cells in G0 + G1 were apparent when fibroblast cells were cultured to confluency or serum starved, and significantly increased (P < 0.01) as the starvation period was prolonged. It is concluded that for particular cell types specific strategies should be used to attain improvements in the efficiency of cloning procedures.

Effect of rabbit age on sperm chromatin structure.

The aim of this study was to determine the relationship between the age of male rabbits and the sperm chromatin structure. The studies involved the semen of New Zealand White rabbits between 5 and 28 months of age. A flow cytometry and sperm chromatin structure assay (SCSA) method was used to determine chromatin structure. The results of cytometric chromatin structure assay suggested a relatively high stability of sperm chromatin in the rabbit. Between 6 and 16 months of age, the mean percentage of sperm with damaged chromatin was the lowest and ranged from 1.7 to 2.4%. Decreased sperm chromatin stability was found in ejaculates taken from male rabbits less than 5 months and more than 20 months of age.

[On the problem of human cloning]. / Wokól problemu konowania czlowieka.

Somatic cell cloning technique in mammals is still not very efficient, but intensive efforts have been made to improve it. Considering the great biological affinity of humans and animals, the cloning technique can in the not too distant future be applied in human cloning and improved to the point of becoming safe. Even when we make such an assumption, I consider it irrational and dangerous to clone the human in order to make their copies (with human cloning for therapeutic purposes being another problem). Life, which is generated by the union of egg cell and spermatozoon is an unforeseeable combination of genetic possibilities, but at the same time it offers a unique chance for the human being, both as an individual and a species. The creation of life by genetic duplication of an already formed individual means a great reduction not only in the biological sense. Action like this is evidence of extreme egocentrism and totalitarian thinking, and its proponents should first answer the question whether they would consider cloning themselves. An answer in the affirmative would help to establish the underlying reasons for their approval.