Transduction profiles in minipig following MRI guided delivery of AAV-5 into thalamic and corona radiata areas.

BACKGROUND: As a step towards clinical use of AAV-mediated gene therapy, brains of large animals are used to settle delivery parameters as most brain connections, and relative sizes in large animals and primates, are reasonably common. Prior to application in the clinic, approaches that have shown to be successful in rodent models are tested in larger animal species, such as dogs, non-human primates, and in this case, minipigs. NEW METHOD: We evaluated alternate delivery routes to target the basal ganglia by injections into the more superficial corona radiata, and, deeper into the brain, the thalamus. Anatomically known connections can be used to predict the expression of the transgene following infusion of AAV5. For optimal control over delivery of the vector with regards to anatomical location in the brain and spread in the tissue, we have used magnetic resonance image-guided convection-enhanced diffusion delivery. RESULTS: While the transduction of the cortex was observed, only partial transduction of the basal ganglia was achieved via the corona radiata. Thalamic administration, on the other hand, resulted in widespread transduction from the midbrain to the frontal cortex COMPARISON WITH EXISTING METHODS: Compared to other methods, such as delivery directly to the striatum, thalamic injection may provide an alternative when for instance, injection into the basal ganglia directly is not feasible. CONCLUSIONS: The study results suggest that thalamic administration of AAV5 has significant potential for indications where the transduction of specific areas of the brain is required.

Minipig model of Huntington's disease: ¹H magnetic resonance spectroscopy of the brain.

Huntington's disease (HD) is an inherited autosomal neurodegenerative disorder affecting predominantly the brain, characterized by motor dysfunctions, behavioral and cognitive disturbances. The aim of this study was to determine changes in the brain of transgenic minipigs before HD onset using (1)H magnetic resonance (MR) spectroscopy. Measurements were performed on a 3 T MR scanner using a single voxel spectroscopy sequence for spectra acquisition in the white matter and chemical shift imaging sequence for measurement in the striatum, hippocampus and thalamus. A decrease of (phospho)creatine (tCr) concentration was found only in the thalamus (p=0.002) of transgenic minipigs, nevertheless we found significant changes in metabolite ratios. Increase of the ratio choline compounds (tCho)/tCr was found in all examined areas: striatum (p=0.010), thalamus (p=0.011) as well as hippocampus (p=0.027). The ratio N-acetylaspartate+N-acetylaspartylglutamate (tNAA)/tCr (p=0.043) and glutamate+glutamine (Glx)/tCr (p=0.039) was elevated in the thalamus, the ratio myo-inositol (Ins)/tCr (p=0.048) was significantly increased in the hippocampus. No significant differences were observed in the metabolite concentrations in the white matter, however we found significant increase of ratios tNAA/tCr (p=0.018) and tCho/tCr (p=0.003) ratios in transgenic boars. We suppose that the majority of the observed changes are predominantly related to changes in energy metabolism caused by decrease of tCr.

Time course of spinal doublecortin expression in developing rat and porcine spinal cord: implication in in vivo neural precursor grafting studies.

Expression of doublecortin (DCX), a 43-53 kDa microtubule binding protein, is frequently used as (i) an early neuronal marker to identify the stage of neuronal maturation of in vivo grafted neuronal precursors (NSCs), and (ii) a neuronal fate marker transiently expressed by immature neurons during development. Reliable identification of the origin of DCX-immunoreactive cells (i.e., host vs. graft) requires detailed spatial and temporal mapping of endogenous DCX expression at graft-targeted brain or spinal cord regions. Accordingly, in the present study, we analyzed (i) the time course of DCX expression in pre- and postnatal rat and porcine spinal cord, and (ii) the DCX expression in spinally grafted porcine-induced pluripotent stem cells (iPS)-derived NSCs and human embryonic stem cell (ES)-derived NSCs. In addition, complementary temporospatial GFAP expression study in porcine spinal cord was also performed. In 21-day-old rat fetuses, an intense DCX immunoreactivity distributed between the dorsal horn (DH) and ventral horn was seen and was still present in the DH neurons on postnatal day 20. In animals older than 8 weeks, no DCX immunoreactivity was seen at any spinal cord laminae. In contrast to rat, in porcine spinal cord (gestational period 113-114 days), DCX was only expressed during the pre-natal period (up to 100 days) but was no longer present in newborn piglets or in adult animals. Immunohistochemical analysis was confirmed with a comparable expression profile by western blot analysis. Contrary, the expression of porcine GFAP started within 70-80 days of the pre-natal period. Spinally grafted porcine iPS-NSCs and human ES-NSCs showed clear DCX expression at 3-4 weeks postgrafting. These data indicate that in spinal grafting studies which employ postnatal or adult porcine models, the expression of DCX can be used as a reliable marker of grafted neurons. In contrast, if grafted neurons are to be analyzed during the first 4 postnatal weeks in the rat spinal cord, additional markers or grafted cell-specific labeling techniques need to be employed to reliably identify grafted early postmitotic neurons and to differentiate the DCX expression from the neurons of the host.

Comparing over-the-scope clip versus endoloop and clips (KING closure) for access site closure: a randomized experimental study.

BACKGROUND: A safe technique is essential for successful access site closure in Natural Orifice Translumenal Endoscopic Surgery (NOTES) and for closures of iatrogenic perforations. AIM: To compare an over-the-scope clip (OTSC) versus an endoloop + endoclips closure technique (KING closure). METHODS: 40 minipigs underwent NOTES peritoneoscopy with liver biopsy. Gastrotomies and rectotomies were closed with OTSC (n = 20; 10× stomach, 10× rectum) or KING closure (n = 20; 10× stomach, 10× rectum). The animals were euthanized 28 days after the procedure. The main outcome variables were technical feasibility, effectiveness, and healing. RESULTS: Stomach: All but one closure (KING) was successfully completed. The times of closure were similar between the techniques. At necropsy, all access sites were healed. In two animals (1× KING, 1× OTSC), an abscess, probably related to the closure technique, was found. Histologically, transmural healing with muscular bridging was observable in nine pigs for KING versus two pigs for OTSC closure (p = 0.003). Inflammation was present in three pigs for KING versus seven pigs for OTSC closure (p = 0.08). Rectum: All closures were successfully completed. The times of closure were similar between the techniques. At necropsy, all closure sites had healed. Transmural healing with muscular bridging was present in nine pigs for KING versus two pigs for OTSC closure (p = 0.003). Inflammation was present in two pigs for KING versus seven pigs for OTSC closure (p = 0.03). In one animal (OTSC), an enterocolic fistula developed in the proximity of the closure site. CONCLUSIONS: OTSC and KING closure are comparable closure techniques in terms of technical feasibility and effectiveness. KING closure provides a superior histological outcome compared with OTSC closure.

Survival and differentiation of human embryonic stem cell-derived neural precursors grafted spinally in spinal ischemia-injured rats or in naive immunosuppressed minipigs: a qualitative and quantitative study.

In previous studies, we have demonstrated that spinal grafting of human or rat fetal spinal neural precursors leads to amelioration of spasticity and improvement in ambulatory function in rats with spinal ischemic injury. In the current study, we characterize the survival and maturation of three different human embryonic stem (ES) cell line-derived neural precursors (hNPCs) once grafted into ischemia-injured lumbar spinal cord in rats or in naive immunosuppressed minipigs. Proliferating HUES-2, HUES-7, or HUES-9 colonies were induced to form embryoid bodies. During the nestin-positive stage, the rosettes were removed and CD184(+)/CD271(-)/CD44(-)/CD24(+) population of ES-hNPCs FAC-sorted and expanded. Male Sprague-Dawley rats with spinal ischemic injury or naive immunosuppressed Gottingen-Minnesota minipigs received 10 bilateral injections of ES-NPCs into the L2-L5 gray matter. After cell grafting, animals survived for 2 weeks to 4.5 months, and the presence of grafted cells was confirmed after staining spinal cord sections with a combination of human-specific (hNUMA, HO14, hNSE, hSYN) or nonspecific (DCX, MAP2, CHAT, GFAP, APC) antibodies. In the majority of grafted animals, hNUMA-positive grafted cells were identified. At 2-4 weeks after grafting, double-labeled hNUMA/DCX-immunoreactive neurons were seen with extensive DCX(+) processes. At survival intervals of 4-8 weeks, hNSE(+) neurons and expression of hSYN was identified. Some hSYN-positive terminals formed putative synapses with the host neurons. Quantitative analysis of hNUMA(+) cells at 2 months after grafting showed comparable cell survival for all three cell lines. In the presence of low-level immunosuppression, no grafted cell survival was seen at 4.5 months after grafting. Spinal grafting of proliferating pluripotent HUES-7 cells led to consistent teratoma formation at 2-6 weeks after cell transplantation. These data show that ES-derived, FAC-sorted NPCs can represent an effective source of human NPCs to be used in CNS cell replacement therapies.

Osteogenic differentiation of miniature pig mesenchymal stem cells in 2D and 3D environment.

Mesenchymal stem cells (MSCs) have been repeatedly shown to be able to repair bone defects. The aim of this study was to characterize the osteogenic differentiation of miniature pig MSCs and markers of this differentiation in vitro. Flow-cytometrically characterized MSCs were seeded on cultivation plastic (collagen I and vitronectin coated/uncoated) or plasma clot (PC)/plasma-alginate clot (PAC) scaffolds and differentiated in osteogenic medium. During three weeks of differentiation, the formation of nodules and deposition of calcium were visualized by Alizarin Red Staining. In addition, the production of alkaline phosphatase (ALP) activity was quantitatively detected by fluorescence. The expression of osteopontin, osteonectin and osteocalcin were assayed by immunohistochemistry and Western Blot analysis. We revealed a decrease of osteopontin expression in 2D and 3D environment during differentiation. The weak initial osteonectin signal, culminating on 7(th) or 14(th) day of differentiation, depends on collagen I and vitronectin coating in 2D system. The highest activity of ALP was detected on 21(th) day of osteogenic differentiation. The PC scaffolds provided better conditions for osteogenic differentiation of MSCs than PAC scaffolds in vitro. We also observed expected effects of collagen I and vitronectin on the acceleration of osteogenic differentiation of miniature pig MSC. Our results indicate similar ability of miniature pig MSCs osteogenic differentiation in 2D and 3D environment, but the expression of osteogenic markers in scaffolds and ECM coated monolayers started earlier than in the monolayers without ECM.

Differential regulation of galectin expression/reactivity during wound healing in porcine skin and in cultures of epidermal cells with functional impact on migration.

The glycophenotyping of mammalian cells with plant lectins maps aspects of the glycomic profile and disease-associated alterations. A salient step toward delineating their functional dimension is the detection of endogenous lectins. They can translate sugar-encoded changes into cellular responses. Among them, the members of the lectin family of galectins are emerging regulators of cell adhesion, migration and proliferation. Focusing on galectins-1, -3 and -7, we addressed the issue whether their expression is regulated during wound healing in porcine skin as model. A conspicuous upregulation is detected for galectin-1 in the dermis and a neoexpression in the epidermis, where an increased level of galectin-7 was also found. Applying biotinylated tissue lectins as probes, the signal intensities for accessible binding sites decreased, intimating an interaction of the cell lectin with reactive sites. In contrast, galectin-3 parameters remained rather constant. Of note, epidermal cells in culture also showed an increase in expression/presence of galectin-1, measured on the levels of mRNA and protein, in this case by Western blotting and quantitative immunocytochemistry. Used as matrix, galectin-1 conferred resistance to trypsin treatment to attached human keratinocytes and reduced migration into scratch-wound areas in vitro. This report thus presents new information on endogenous lectins in wound healing and differential regulation among the three tested cases.

Composite hyaluronate-type I collagen-fibrin scaffold in the therapy of osteochondral defects in miniature pigs.

The potential of novel scaffold containing sodium hyaluronate, type I collagen, and fibrin was investigated in the regeneration of osteochondral defects in miniature pigs. Both autologous chondrocyte-seeded scaffolds and non-seeded scaffolds were implanted into two defects located in the non-weight-bearing zone of the femoral trochlea (defect A was located more distally and medially, defect B was located more proximally and laterally). Control defects were left untreated. Twelve weeks after the operation, the knees were evaluated in vivo using MRI. Six months after the implantation, the defects were analyzed using MRI, histological, and immunohistochemical analysis. In the A defects of chondrocyte-seeded scaffold group, hyaline cartilage and fibrocartilage was formed, containing type II collagen, acidic and neutral glycosaminoglycans while the non-seeded scaffold group was predominantly filled with fibrocartilage. Defects in the control group were predominantly filled with fibrous tissue. Histomorphometric analysis of photomicrographs revealed a significantly higher amount of hyaline cartilage in the cell-seeded scaffold group in A defects than in other groups. Both scaffold groups in A defects showed significantly less fibrous tissue than cell-seeded defects B and the control group. Both histological and MRI analysis proved that the novel composite scaffold has a potential to regenerate osteochondral defects within six months.

Phenotypic characterization of porcine interfollicular keratinocytes separated by elutriation: a technical note.

Separation of epidermal stem cells from other populations in suspensions of epidermal cells by sorting is hampered by a present lack of specific surface markers of this cell type. To address this issue we applied CCE combined with immunocytochemical phenotyping. On the basis of expression profiles for keratins (10, 14, and 19), nucleostemin, galectin-1 and epitopes reactive for this adhesion/growth-regulatory tissue lectin we identified a fraction of very small cells originating from the basal layer. The results demonstrated that CCE has potential merit for separation of epidermal cells to yield a population likely enriched in stem cells.

Porcine epidermal stem cells as a biomedical model for wound healing and normal/malignant epithelial cell propagation.

This article summarizes research using cells derived from epidermis of the miniature pigs for use as a cell therapy for skin repair and as a model for squamous carcinoma of the head and neck. Stem cells are an important "tool" for biomedical research. Adult stem cells are defined functionally, as cells that have the capacity to self-renew as well as the ability to generate differentiated cells. They are present in defined tissue microenvironments called niches. Asymmetric mitosis allows them to produce one daughter cell with the properties of stem cells (self-renewal) and a second cell with characteristics of progenitor cells, or transit amplifying cells, which proliferate quickly but with a limited number of mitotic divisions. Porcine epidermal stem cells, located in the bulge region of the outer root sheath of hair follicles, migrate in vitro from hair sheaths and because they are resistant to anoikis (detachment induced apoptosis), survive in non-adhesive conditions to form spheroids. These cells express keratins, galectin-1 and their nuclei are rich in DeltaNp63alpha. Interestingly, the multiple phenotype analysis of the human tumor cells in squamous carcinoma of head and neck revealed similarities with epidermal stem cells. These cancer stem cells are usually located on the periphery of the tumor where the invasive front of the tumor responsible for its aggressive behavior is located. In contrast, extensive expression of markers of terminal differentiation such as expression of glycoligands reactive for the endogenous lectin, galectin-3, indicates better tumor prognosis.

Development of in vivo derived diploid and tetraploid pig embryos in a modified medium NCSU 37.

The aim of this study was to assess development of diploid and tetraploid in vivo derived pig embryos cultured in a modified medium NCSU 37 in an atmosphere with reduced concentration of oxygen. The tetraploid embryos were produced by electrofusion of two-cell embryos that had been cultured in vitro from the one-cell stage before fusion (cultured two-cell embryos) or by fusion of freshly recovered two-cell embryos. Development to blastocyst stage of tetraploid embryos, generated from the cultured two-cell embryos was significantly inferior to the development of control one-cell embryos (29.1 +/- 9.7% versus 66.8 +/- 9.7%; P < 0.05). However, development of tetraploid embryos produced from the freshly recovered two-cell embryos and control two-cell embryos was very similar (89.9 +/- 6.1% versus 81.3 +/- 3.4%). Detection of chromosomes 1 and 10 by in situ hybridization showed that more than 85% of the cultured control embryos were diploid while 15% of the embryos were mosaic. Among the fused embryos 50% were tetraploid, 29% mosaic and 21% diploid. These data indicate that the modified medium NCSU 37 provides optimum environment for pre-implantation development of pig diploid and tetraploid embryos.

Mammalian oocyte activation: lessons from the sperm and implications for nuclear transfer.

Events after fertilisation have been carefully studied in the last decades. However, there are still several questions to be clarified in relation to the signalling pathway initiated by the sperm, the identification of proteins or factors involved in the activation of the arrested oocyte, and the inactivation of specific molecules involved in the meiotic arrest. The present state of knowledge in mammalian fertilisation allows the development of activation protocols that closely mimic the events initiated by the sperm according to certain major factors (MPF activity and MAPk activity). These protocols are successfully used for the activation of oocytes after NT giving rise to offspring. Few cloned animals have yet been produced. However, the pregnancy and the survival rates after birth are not significantly different when different activation protocols are compared. This fact argues fora major reason forthe low success in the efficiency of NT. Eventually, factors related to the recipient oocyte, the donor cell or the culture conditions are part of these major problems that the reconstructed embryo has to overcome to develop into a normal offspring. Nonetheless, the development of activation protocols that closely imitate the mechanism of activation initiated by the sperm are of special interest to improve the developmental potential of cloned embryos.

Remodeling of donor nuclei, DNA-synthesis, and ploidy of bovine cumulus cell nuclear transfer embryos: effect of activation protocol.

The purpose of this study was to investigate the effects of two activation protocols on nuclear remodeling, DNA synthesis during the first cell cycle, chromosome segregation after first mitosis and development to blastocyst of embryos produced by somatic nuclear transfer. Pronuclear formation was significantly higher when activation lasted 5 hr compared to 3 hr for both ethanol-cycloheximide and ionomycin-bohemine treatment. However, the presence of a single nucleus was significantly higher in embryos activated for 3 hr in bohemine. Initiation of DNA synthesis was delayed in ethanol-cycloheximide group, however, after 12 hr labeling 100% of embryos synthesized DNA in both groups. Embryos activated with ethanol-cycloheximide developed to blastocysts at a significantly higher rate than those activated with ionomycin-bohemine. Analysis of 2-cell embryos with DNA probes for chromosome 6, 7, and 15 by fluorescence in situ hybridization showed that at least 50% of NT embryos were of normal ploidy independent of the activation stimulus. The results presented in this study show differences between the protocols compared on the nuclear events during the first cell cycle and on the development to blastocyst. Mol. Reprod. Dev. 59: 371-379, 2001.

Insulin-like growth factor I (IGF-I) and long R(3)IGF-I differently affect development and messenger ribonucleic acid abundance for IGF-binding proteins and type I IGF receptors in in vitro produced bovine embryos.

The insulin-like growth factor (IGF) system is a complex network, including ligands (IGF-I and -II), binding proteins (IGFBP-1 to -6), and receptors, of which the type I IGF receptor (IGF-I-R) is important for transmission of most biological effects of IGFs. As IGFs are secreted in large amounts by the female reproductive tract, it has been hypothesized that maternal IGFs may affect embryonic growth and differentiation in a fine-tuned manner, involving modulation of IGF effects by embryonic IGFBP and IGF-I-R expression. To address this point, we cultured in vitro produced bovine embryos in a chemically defined culture system in the presence (100 ng/ml) of recombinant human IGF-I, long R(3)IGF-I (LR(3)), or without IGF supplementation (control). The affinity of LR(3) to IGFBPs measured by competition assays and Western ligand blots is at least 3 orders of magnitude lower than that of IGF-I. LR(3) was most efficient in stimulating early embryonic cleavage, whereas further development was most potently supported by IGF-I. Total cell numbers of blastocysts were highest in the presence of LR(3) (105 +/- 4), followed by IGF-I (96 +/- 5), and the control group (91 +/- 3; P < 0.05). Differential cell staining of blastocysts revealed that these differences were mainly represented by trophectoderm cell numbers. Analysis of messenger RNA (mRNA) expression for IGFBPs and IGF-I-R was performed by RT-real-time PCR, using expression of the nonregulated housekeeping gene glyceraldehyde-3-phosphate dehydrogenase for normalization. Embryonic IGFBP-2 mRNA levels in the LR(3) treatment group were 1.7-fold (P < 0.001) and 2.8-fold (P < 0.001) higher than those in the IGF-I and control groups, respectively. IGFBP-5 mRNA levels were about 2-fold (P < 0.001) elevated in both IGF treatment groups, with slightly (P < 0.05) higher levels in IGF-I- than in LR(3)-treated embryos. Similarly, IGFBP-3 mRNA abundance was increased (P < 0.05) in embryos from the IGF-I vs. the LR(3) culture system. IGF-I-R mRNA levels were reduced by IGF-I (80% of control; P < 0.01), but increased by LR(3) (1.3-fold vs. control; P < 0.001). These data show that the affinity for IGFBPs of IGF peptides is relevant for their effects on preimplantation embryos and affects different parameters, i.e. development, cell numbers, and mRNA expression for components of the IGF system, in different directions.

Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts.

Ubiquitination is a universal protein degradation pathway in which the molecules of 8.5-kDa proteolytic peptide ubiquitin are covalently attached to the epsilon-amino group of the substrate's lysine residues. Little is known about the importance of this highly conserved mechanism for protein recycling in mammalian gametogenesis and fertilization. The data obtained by the students and faculty of the international training course Window to the Zygote 2000 demonstrate the accumulation of ubiquitin-cross-reactive structures in the trophoblast, but not in the inner cell mass of the expanding bovine and mouse blastocysts. This observation suggests that a major burst of ubiquitin-dependent proteolysis occurs in the trophoblast of mammalian peri-implantation embryos. This event may be important for the success of blastocyst hatching, differentiation of embryonic stem cells into soma and germ line, and/or implantation in both naturally conceived and reconstructed mammalian embryos.

Culture of bovine oocytes from small antral follicles in meiosis-inhibiting medium with butyrolactone I: RNA synthesis, nucleolar morphology and meiotic competence.

Bovine oocytes originated from follicles of two different size categories (medium (M), 3-6 mm and small (S), 1-2 mm) were cultured for 24 and 70 h, respectively, in a meiosis-inhibiting medium (MIM) supplemented with 100 microM butyrolactone I (BL I). At the end of culture, cumulus oocyte complexes (COC) from M and S follicles were labeled by 3H-uridine for 30 min. The autoradiography (ARG) of semi-thin sections of COC showed the labeling of the germinal vesicles (GV) as: (1) The COC of the M category labeled immediately after isolation from follicles showed only weak labeling (+) of the GV. The COC of the S category labeled immediately after their isolation showed mostly intensive labeling (4+,3+) of the GV. (2) When the COC were labeled after 24 and 70 h of culture in MIM, no labeling was observed in the M category. The S category of oocytes showed the slightly decreased labeling (3+,2+) after 24 h and negligible labeling after 70 h of culture. The pattern of very intensive labeling of granulosa cell nuclei of all mentioned groups was practically not changed during the whole 70 h period of culture in both categories. The nucleolar ultrastructure of S category oocytes revealed time dependent changes from the reticular fibrillogranular structure present in freshly isolated oocytes. The several fibrillar centers before the culture changed to the fibrillogranular appearance with few large and a number of small vacuoles and an exclusively fibrillar area after 24 h of culture. Finally, nucleoli acquired a mostly exclusively fibrillar structure with one large fibrillar center after 70 h of culture. In the second experiment, the meiotic maturation of COC of S category was inhibited in MIM for 48 h. The subsequent 24 h culture in a medium with BOS and gonadotropins resulted in 81.0% oocytes matured to metaphase II (M II). Only 27.1 and 11.3% of the control S oocytes cultured in a medium, with BOS and gonadotropins directly after isolation, reached M II after 48 or 72 h of culture, respectively. The two-step culture increased significantly the meiotic competence of cattle oocytes isolated from small antral follicles.

Behavior of M-phase synchronized blastomeres after nuclear transfer in cattle.

M-phase synchronized bovine blastomeres were used to study the effect of nuclear-cytoplasmic synchronization on the developmental potential after nuclear transfer (NT). The capacity of nocodazole and benomyl to reversibly synchronize blastomeres from embryos in M-phase was evaluated. Nocodazole reversibly arrested bovine embryos at the studied stages and induced high rates of M-phases in morulae and compact morulae. In contrast, benomyl was less efficient than nocodazole to synchronize in M-phase. After transfer of an M-phase blastomere, premature chromatin condensation was the prevalent finding 1 hr post-fusion (hpf). Condensed chromosomes non-arranged in the equatorial plate (1-3 hpf) that acquired an organized structure over time (3-7 hpf) were subsequently observed. Anaphase-telophase structures were predominantly recorded at 4-9 hpf. About 50% of the embryos activated at both 3-4 and 6-7 hpf extruded a polar body-like structure 5 hr after activation, but this was not observed in embryos activated immediately after fusion. A significantly lower activation rate was observed for oocytes activated 3-4 hpf compared to those activated 6-7 hpf. However, the ability to undergo first cleavage was significantly lower in the latter group. Reconstructed embryos activated immediately after fusion showed no difference in the rate of activation compared to those activated 6-7 hpf, although the cleavage rate was higher. DNA synthesis was observed at a significantly higher rate in embryos activated both immediately and 3-4 hpf that did not extrude a PB-like structure than in those activated 3-4 hpf that extruded a polar body-like structure. Under the conditions tested M-phase donor cells cannot be properly remodeled after NT in cattle to trigger normal embryonic development. Our observations of chromatin structures together with DNA synthesis suggest that the failure in the development may be due to improper chromatin remodeling of mitotic nuclei after NT, which may result in chromosomal abnormalities incompatible with normal embryo development.

Activation of bovine oocytes by specific inhibition of cyclin-dependent kinases.

Activation of bovine oocytes by experimental procedures that closely mimic normal fertilization and allow to obtain haploid oocytes is essential both for intracytoplasmic sperm injection (ICSI) and for nuclear transfer. Therefore, with the goal of producing haploid activated oocytes, this study evaluated whether bohemine, either alone or in combination with ionomycin, is able to activate young matured bovine oocytes. Furthermore, the effect of bohemine on the patterns of DNA synthesis after pronuclear formation as well as changes in histone H1 kinase and MAP kinase activities during the process of activation were studied. Our results with bohemine show that the specific inhibition of CDKs in metaphase II bovine oocytes induces parthenogenetic activation in a dose-dependent manner (25, 50, and 100 microM, respectively), either alone (3%, 30%, and 50%) or in combination with ionomycin (30%, 70%, and 87.5%). A single pronucleus and extrusion of the second polar body was observed (97%) when Ca(2+) influx was stimulated in the presence of bohemine, although pronuclear formation without polar body extrusion was observed when bohemine was used alone. Bohemine-activated oocytes started to synthesize DNA in the first hour (37%) after their removal from bohemine-supplemented medium (6-7 hr post-activation; hpa). A high synchrony in the S-phase was registered with more than 85% of parthenotes actively synthesizing DNA 8 hpa. By contrast, DNA synthesis was absent in oocytes cultured for 4, 6, and 8 hpa in the presence of bohemine and a low rate was observed by those cultured for 18 hr (30%) in bohemine-supplemented medium. This confirms the ability of the inhibitor to arrest the cell cycle in the G1/S boundary for at least 8 hr. A drop in histone H1 kinase activity was observed in bohemine-activated oocytes. The activity of MBP kinase decreased later than histone H1 kinase and even 4 hr after inomycin-bohemine treatment at least half of this activity was still detectable. Then, the MBP kinase activity decreased and the lowest level could be seen 6-8 hpa. In summary, our study shows that in vitro matured bovine oocytes can be successfully activated by a synthetic inhibitor of CDKs. This effect can be improved by combination with ionomycin. The targeting of CDKs in the way to activate bovine oocytes can be an approach to improve the efficiency of mammalian oocyte activation.

Butyrolactone I reversibly inhibits meiotic maturation of bovine oocytes,Without influencing chromosome condensation activity.

In this study, butyrolactone I (BL I), a potent and specific inhibitor of cyclin-dependent kinases, was shown to block germinal vesicle (GV) breakdown (GVBD) in bovine oocytes in a concentration-dependent manner; GVBD was almost totally inhibited over the course of 24-48 h of culture when 100 microM BL I was included in tissue culture medium 199 containing either polyvinyl alcohol or BSA. Correlated with this inhibition was the failure of either p34(cdc2) kinase or mitogen-activated protein (MAP) kinase to become activated, and it was unlikely that BL I directly inhibited MAP kinase, since 100 microM BL I did not inhibit MAP kinase activity present in extracts obtained from metaphase II-arrested bovine eggs that possess high levels of MAP kinase activity. Nevertheless, the formation of highly condensed bivalents was observed in 78% of the BL I-treated GV-intact oocytes. This result suggests that chromosome condensation during first meiosis in bovine oocytes does not require the activity of either p34(cdc2) kinase or MAP kinase. Treatment of BL I-arrested oocytes with okadaic acid (OA) did not result in either the activation of p34(cdc2) kinase or MAP kinase, or inducement of GVBD. The BL I-induced block of GVBD for 24 h was reversible, and a subsequent 24-h culture resulted in 90% of oocytes reaching metaphase II with emission of the first polar body. Correlated with the progression to and arrest at metaphase II was the full activation of both p34(cdc2) and MAP kinases. The reversibility after 48 h of culture in BL I was partially decreased when compared to that achieved after an initial 24-h culture. Fertilization in vitro of these eggs resulted in a high incidence of both sperm penetration and pronucleus formation (88% and 70%, respectively).

Cell cycle synchronization of porcine fetal fibroblasts: effects of serum deprivation and reversible cell cycle inhibitors.

The success of somatic nuclear transfer critically depends on the cell cycle stage of the donor nucleus and the recipient cytoplast. In this study we tested serum deprivation as well as two reversible cell cycle inhibitors, aphidicolin and butyrolactone I, for their ability to synchronize porcine fetal fibroblasts at either G0 stage or G1/S or G2/M transition. The synchronization efficiency of the various protocols was determined by fluorescence-activated cell sorting (FACS), cell proliferation assays, and semiquantitative multiplex reverse transcription-polymerase chain reaction detection of the cell cycle-regulated porcine Polo-like kinase mRNA (Plk-p). FACS measurements revealed that 66.6-73.3% of the porcine fetal fibroblasts were in G0/G1 stage (2C DNA content) in serum-supplemented medium. Short periods of 24-72 h of serum deprivation significantly increased the proportion of cells at G0/G1 phase to 77.9-80.2%, and mitotic activity had already terminated after 48 h. Prolonged culture in serum-deprived medium induced massive DNA fragmentation. Aphidicolin treatment led to an accumulation of 81.9 +/- 4.9% of cells at the G1/S transition. Butyrolactone I arrested 81.0 +/- 5.8% of the cells at the end of G1 stage and 37.0 +/- 6.8% at the G2/M transition. The effects of both chemical inhibitors were fully reversible, and their removal led to a rapid progression in the cell cycle. The measurement of Plk-p expression allowed discrimination between the presumptive G0 phase induced by serum deprivation and the G1/S transition arrest achieved by chemical inhibitors. These data indicate that porcine fetal fibroblasts can be effectively synchronized at various cell cycle stages without compromising their proliferation capacity.