Jan Motlik

The Miniature Pig as an Animal Model in Biomedical Research

Abstract: Crucial prerequisites for the development of safe preclinical protocols in biomedical research are suitable animal models that would allow for human‐related validation of valuable research information gathered from experimentation with lower mammals. In this sense, the miniature pig, sharing many physiological similarities with humans, offers several breeding and handling advantages (when compared to non‐human primates), making it an optimal species for preclinical experimentation. The present review offers several examples taken from current research in the hope of convincing the reader that the porcine animal model has gained massively in importance in biomedical research during the last few years. The adduced examples are taken from the following fields of investigation: (a) the physiology of reproduction, where pig oocytes are being used to study chromosomal abnormalities (aneuploidy) in the adult human oocyte; (b) the generation of suitable organs for xenotransplantation using transgene expression in pig tissues; (c) the skin physiology and the treatment of skin defects using cell therapy‐based approaches that take advantage of similarities between pig and human epidermis; and (d) neurotransplantation using porcine neural stem cells grafted into inbred miniature pigs as an alternative model to non‐human primates xenografted with human cells.

Cell Cycle Synchronization of Porcine Fetal Fibroblasts: Effects of Serum Deprivation and Reversible Cell Cycle Inhibitors

Abstract 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.

Mapping of the secretome of primary isolates of mammalian cells, stem cells and derived cell lines.

Within a mammalian organism, the interaction among cells both at short and long distances is mediated by soluble factors released by cells into the extracellular environment. The secreted proteins may involve extracellular matrix proteins, proteinases, growth factors, protein hormones, immunoregulatory cytokines, chemokines or other bioactive molecules that have a direct impact on target cell phenotype. Stem cells of mesenchymal, adipose, neural and embryonic origin, fibroblast feeder cells as well as primary isolates of astrocytes, endothelial and muscle cells have recently become targets of intensive secretome profiling with the search for proteins regulating cell survival, proliferation, differentiation or inflammatory response. Recent advances and challenges of the stem cell and primary cell secretome analysis together with the most relevant results are discussed in this review.

Butyrolactone I Reversibly Inhibits Meiotic Maturation of Bovine Oocytes,Without Influencing Chromosome Condensation Activity

Abstract 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 μM BL I was included in tissue culture medium 199 containing either polyvinyl alcohol or BSA. Correlated with this inhibition was the failure of either p34cdc2 kinase or mitogen-activated protein (MAP) kinase to become activated, and it was unlikely that BL I directly inhibited MAP kinase, since 100 μM 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 p34cdc2 kinase or MAP kinase. Treatment of BL I-arrested oocytes with okadaic acid (OA) did not result in either the activation of p34cdc2 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 p34cdc2 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).

Prophase I arrest and progression to metaphase I in mouse oocytes: comparison of resumption of meiosis and recovery from G2-arrest in somatic cells

Mammalian oocytes are arrested at prophase I until puberty when luteinizing hormone (LH) induces resumption of meiosis of follicle-enclosed oocytes. Resumption of meiosis is tightly coupled with regulating cyclin-dependent kinase 1 (CDK1) activity. Prophase I arrest depends on inhibitory phosphorylation of CDK1 and anaphase-promoting complex-(APC-CDH1)-mediated regulation of cyclin B levels. Prophase I arrest is maintained by endogenously produced cyclic adenosine monophosphate (cAMP), which activates protein kinase A (PKA) that in turn phosphorylates (and activates) the nuclear kinase WEE2. In addition, PKA-mediated phosphorylation of the phosphatase CDC25B results in its cytoplasmic retention. The combined effect maintains low levels of CDK1 activity that are not sufficient to initiate resumption of meiosis. LH triggers synthesis of epidermal growth factor-like factors in mural granulosa cells and leads to reduced cGMP transfer from cumulus cells to oocytes via gap junctions that couple the two cell types. cGMP inhibits oocyte phosphodiesterase 3A (PDE3A) and a decline in oocyte cGMP results in increased PDE3A activity. The ensuing decrease in oocyte cAMP triggers maturation by alleviating the aforementioned phosphorylations of WEE2 and CDC25B. As a direct consequence CDC25B translocates into the nucleus. The resulting activation of CDK1 also promotes extrusion of WEE2 from the nucleus thereby providing a positive amplification mechanism for CDK1 activation. Other kinases, e.g. protein kinase B, Aurora kinase A and polo-like kinase 1, also participate in resumption of meiosis. Mechanisms governing meiotic prophase I arrest and resumption of meiosis share common features with DNA damage-induced mitotic G2-checkpoint arrest and checkpoint recovery, respectively. These common features include CDC14B-dependent activation of APC-CDH1 in prophase I arrested oocytes or G2-arrested somatic cells, and CDC25B-dependent cell cycle resumption in both oocytes and somatic cells.

PKB/AKT is involved in resumption of meiosis in mouse oocytes

Background information. In fully grown mouse oocytes, a decrease in cAMP concentration precedes and is linked to CDK1 (cyclin‐dependent kinase 1) activation. The molecular mechanism for this coupling, however, is not defined. PKB (protein kinase B, also called AKT) is implicated in CDK1 activation in lower species. During resumption of meiosis in starfish oocytes, MYT1, a negative regulator of CDK1, is phosphorylated by PKB in an inhibitory manner. It can imply that PKB is also involved in CDK1 activation in mammalian oocytes.

Protein Patterns of Pig Oocytes During In Vitro Maturation

Abstract In vitro maturation (IVM) of fully grown mammalian oocytes is characterized by initial germinal vesicle (GV) breakdown and rearrangement of microtubule network during the first meiosis (MI), followed by extrusion of the first polar body and block of the oocytes in metaphase of the second meiosis (MII). Only fully matured oocytes are capable of undergoing fertilization and the initiation of zygotic development. These observations are mostly based on morphological evaluation; however, the molecular events responsible for these processes are not known. In this study, we have launched the analysis of pig oocytes during in vitro maturation using a proteomics approach. First, oocyte proteins have been separated by two-dimensional gel electrophoresis and identified by mass spectrometry. Remarkably, several proteins, including peroxiredoxins, ubiquitin carboxyl-terminal hydrolase isozyme L1, and spermine synthase, are even more abundant than actin, usually the most abundant protein in somatic cells. Furthermore, we have initiated comparative analysis of the oocytes at different stages of maturation to characterize candidate proteins, which are differentially expressed during in vitro maturation. To date, we have identified antiquitin (D7A1), the member of aldehyde dehydrogenase family7 that has been significantly increased in MI and MII stages compared with GV oocytes. To our knowledge, this is the first pig oocyte proteome available so far that may be used as a reference map. The proteins that are differentially regulated during IVM may present potential biomarkers of oocyte maturation and quality. It is a useful inventory toward a deeper understanding of the mechanisms underlying reproduction and development.

Aurora kinase A controls meiosis I progression in mouse oocytes

Aurora kinase A (AURKA), which is a centrosome-localized serine/threonine kinase crucial for cell cycle control, is critically involved in centrosome maturation and spindle assembly in somatic cells. Active T288 phosphorylated AURKA localizes to the centrosome in the late G2 and also spreads to the minus ends of mitotic spindle microtubules. AURKA activates centrosomal CDC25B and recruits cyclin B1 to centrosomes. We report here functions for AURKA in meiotic maturation of mouse oocytes, which is a model system to study the G2 to M transition. Whereas AURKA is present throughout the entire GV-stage oocyte with a clear accumulation on microtubule organizing centers (MTOC), active AURKA becomes entirely localized to MTOCs shortly before germinal vesicle breakdown. In contrast to somatic cells in which active AURKA is present at the centrosomes and minus ends of microtubules, active AURKA is mainly located on MTOCs at metaphase I (MI) in oocytes. Inhibitor studies using Roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor) reveal that activation of AURKA localized on MTOCs is independent on PI3K-PKB and CDK1 signaling pathways and MOTC amplification is observed in roscovitine- and SH-6- treated oocytes that fail to undergo nuclear envelope breakdown. Moreover, microinjection of Aurka mRNA into GV-stage oocytes cultured in 3-isobutyl-1-methyl xanthine (IBMX)-containing medium to prevent maturation also results in MOTC amplification in the absence of CDK1 activation. Over-expression of AURKA also leads to formation of an abnormal MI spindle, whereas RNAi-mediated reduction of AURKA interferes with resumption of meiosis and spindle assembly. Results of these experiments indicate that AURKA is a critical MTOC-associated component involved in resumption of meiosis, MTOC multiplication, proper spindle formation and the metaphase I-metaphase II transition.

CDC25A phosphatase controls meiosis I progression in mouse oocytes

CDK1 is a pivotal regulator of resumption of meiosis and meiotic maturation of oocytes. CDC25A/B/C are dual-specificity phosphatases and activate cyclin-dependent kinases (CDKs). Although CDC25C is not essential for either mitotic or meiotic cell cycle regulation, CDC25B is essential for CDK1 activation during resumption of meiosis. Cdc25a -/- mice are embryonic lethal and therefore a role for CDC25A in meiosis is unknown. We report that activation of CDK1 results in a maturation-associated decrease in the amount of CDC25A protein, but not Cdc25a mRNA, such that little CDC25A is present by metaphase I. In addition, expression of exogenous CDC25A overcomes cAMP-mediated maintenance of meiotic arrest. Microinjection of Gfp-Cdc25a and Gpf-Cdc25b mRNAs constructs reveals that CDC25A is exclusively localized to the nucleus prior to nuclear envelope breakdown (NEBD). In contrast, CDC25B localizes to cytoplasm in GV-intact oocytes and translocates to the nucleus shortly before NEBD. Over-expressing GFP-CDC25A, which compensates for the normal maturation-associated decrease in CDC25A, blocks meiotic maturation at MI. This MI block is characterized by defects in chromosome congression and spindle formation and a transient reduction in both CDK1 and MAPK activities. Lastly, RNAi-mediated reduction of CDC25A results in fewer oocytes resuming meiosis and reaching MII. These data demonstrate that CDC25A behaves differently during female meiosis than during mitosis, and moreover, that CDC25A has a function in resumption of meiosis, MI spindle formation and the MI-MII transition. Thus, both CDC25A and CDC25B are critical for meiotic maturation of oocytes.

Insulin-Like Growth Factor I (IGF-I) and Long R3IGF-I Differently Affect Development and Messenger Ribonucleic Acid Abundance for IGF-Binding Proteins and Type I IGF Receptors inin VitroProduced Bovine Embryos1

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 R3IGF-I (LR3), or without IGF supplementation (control). The affinity of LR3 to IGFBPs measured by competition assays and Western ligand blots is at least 3 orders of magnitude lower than that of IGF-I. LR3 was most efficient in stimulating early embryonic cleavage, whereas further development was most potently supported by ...