Effect of Follicle Size on In Vitro Maturation of Pre-Pubertal Porcine Cumulus Oocyte Complexes.

Very small follicles (<3.0 mm diameter) are over-represented on the surface of ovaries of non-cycling pigs, and the oocytes collected from these follicles generally have reduced developmental competence in vitro. This study examined the effect of follicle size on the nuclear maturation (n = 608), the potential of parthenogenetic activation (n = 243) and the cyclic AMP (cAMP) content of pre-pubertal porcine oocytes (n = 480). In addition, the influence of follicle size on steroid hormone synthesis was analysed. Cumulus oocyte complexes (COCs) flushed from small (2.5-4.0 mm) or large (4.5-6.0 mm) ovarian follicles were cultured for 0, 28 and 46 h. After 46 h of IVM, a greater proportion of oocytes from 4.5- to 6.0-mm follicles reach metaphase II (MII) compared with those from follicles with 2.5-4.0 mm of diameter (96.1 vs 77.0%, respectively; p < 0.001). Parthenogenetic activation of oocytes from large follicles produced higher developmental rates than oocytes from large follicles (p < 0.05). At 28 h, the IVM medium with oocytes from large follicles contained significantly more 17ß-oestradiol (E2 ) than the medium with oocytes from small follicles (5.55 vs 3.45 ng/ml, respectively; p < 0.05) and at 46 h, the medium with oocytes from small follicles contained significantly more progesterone (P4 ) than the medium with oocytes from large follicles (276.7 vs 108.2 ng/ml, respectively, p < 0.05). Porcine oocytes from large follicles have higher nuclear and cytoplasmic maturation capacities, but the differences did not appear to be cAMP-mediated. Our findings also suggest that COCs from small follicles undergo more intensive luteinization than COCs from large follicles. The results show that oocytes from follicles with a diameter greater than 4.0 mm are more suitable for in vitro studies.

Bone morphogenetic protein-6 (BMP-6): mRNA expression and effect on steroidogenesis during in vitro maturation of porcine cumulus oocyte complexes.

Oocyte secreted factors (OSFs) have emerged as important factors for follicular development. The present study investigated the effect of the potential OSF bone morphogenic protein (BMP)-6 on steroidogenesis in porcine cumulus oocyte complexes during in vitro maturation. Cumulus oocyte complexes (COCs), cumulus complexes (CCs) without oocytes and CCs with supplemented BMP-6 were cultured for 0, 5, 26 or 46 h. BMP-6 transcripts were detected in oocytes and cumulus cells at all time points. In both cell types the mRNA expression was most intense after 5h, and decreased during further maturation. After 26 and 46 h of culture, CCs secreted significantly less 17ß-estradiol than COCs. This effect was reversed by adding BMP-6 to CCs cultures. In addition, a down-regulation of Cyp19A1, the rate-limiting enzyme of 17ß-estradiol synthesis, was detected in CC cultures after 5h. As seen for 17ß-estradiol secretion, the addition of BMP-6 caused a significant increase in Cyp19A1 mRNA levels after 5, 26 and 46 h of culture. Progesterone secretion and transcripts of steroidogenic marker proteins StAR and 3ß-HSD were not affected considerably by oocyte removal or addition of BMP-6. Furthermore, BMP-6 did not affect the activity of the mitogen-activated protein kinase. The results indicated that BMP-6 is a potential OSF and is involved in the prevention of premature luteinisation in cumulus cells via enhancing 17ß-estradiol synthesis.

Steroidogenesis and the influence of MAPK activity during in vitro maturation of porcine cumulus oocyte complexes.

The aim of this study was to investigate steroidogenesis within porcine cumulus oocyte complexes during in vitro maturation and to examine the possible influence of the mitogen-activated protein kinase (MAPK). Porcine cumulus oocyte complexes were matured in vitro with and without the MAPK kinase inhibitor U0126 for 0, 5, 26 and 46 h. The 17ß-estradiol and progesterone concentration in the culture medium were then determined. In addition, the mRNA levels of StAR, Cyp11A1, 3ß-HSD and Cyp19A1 in cumulus cells were analysed by RT-PCR. Using an immunoblot, the MAPK phosphorylation in cumulus cells and oocytes was examined. During the first 26 h of in vitro maturation, 17ß-estradiol secretion was predominant, whereas, after a culture period of 46 h, the progesterone secretion decreased conspicuously. Under the influence of U0126, the secretion of 17ß-estradiol increased progressively during the complete maturation period, while progesterone secretion was completely inhibited. The mRNA levels of StAR and Cyp11A1 were not altered by U0126; however, corresponding to the hormone secretion, the gene expression of Cyp19A1 was up-regulated and the expression of 3ß-HSD down-regulated. The results suggested an influence of the MAPK on steroidogenesis in cumulus cells comparable to a luteinization factor. Hormone synthesis in cumulus cells during oocyte maturation seems to be regulated by altering expression of Cyp19A1 and 3ß-HSD.

Development of an interleukin (IL) 6 receptor antagonist that inhibits IL-6-dependent growth of human myeloma cells.

The pleiotropic cytokine interleukin 6 (IL-6) plays a role in the pathogenesis of various diseases, such as multiple myeloma, autoimmune and inflammatory diseases and osteoporosis. Therefore, specific inhibitors of IL-6 may have clinical applications. We previously succeeded in developing receptor antagonists of IL-6 that antagonized wild-type IL-6 activity on the human Epstein-Barr virus (EBV)-transformed B cell line CESS and the human hepatoma cell line HepG2. However, these proteins still had agonistic activity on the human myeloma cell line XG-1. We here report the construction of a novel mutant protein of IL-6 in which two different mutations are combined that individually disrupt the association of the IL-6/IL-6 receptor (R) alpha complex with the signaltransducing "beta" chain, gp130, but leave the binding of IL-6 to IL-6R alpha intact. The resulting mutant protein (with substitutions of residues Gln160 to Glu, Thr163 to Pro, and replacement of human residues Lys42-Ala57 with the corresponding residues of mouse IL-6) was inactive on XG-1 cells and weakly antagonized wild-type IL-6 activity on these cells. By introducing two additional substitutions (Phe171Leu, Ser177Arg), the affinity of the mutant protein for IL-6R alpha was increased fivefold, rendering it capable of completely inhibiting wild-type IL-6 activity on XG-1 cells. Moreover, this mutant also antagonized the activity of IL-6, but not that of leukemia inhibitory factor, oncostatin M, or GM-CSF on the human erythroleukemia cell line TF-1, demonstrating its specificity for IL-6. These data demonstrate the feasibility of developing specific IL-6R antagonists. The availability of such antagonists may offer an approach to specifically inhibit IL-6 activity in vivo.

In vitro ageing of porcine oocytes: changes in phosphorylation of the mitogen-activated protein kinase (MAPK) and parthenogenetic activability.

The role of mitogen-activated protein kinase (MAPK) was investigated during ageing of porcine oocytes following in vitro maturation (IVM). Oocytes exhibiting an extruded first polar body after IVM for 46 h (79.3% metaphase II, M II) were used for the experiments. Nuclear maturation stages were not visibly altered after a further 12 h of ageing. Proportion of M II stages (42.9%) decreased significantly whereas fragmentation and degeneration of oocytes increased after an ageing time of 26 h. In vitro ageing for 12 and 26 h led to a significant reduction of MAPK phosphorylation (i.e. activation) compared to oocytes matured for 46 h. When MAPK was inhibited by U0126 in M II oocytes, 30.9% (12 h) and 39.7% (26 h) of oocytes, respectively, left metaphase II arrest and proceeded to early anaphase II. Pronuclear stages or fragmentation could be observed only sporadically (2.6-3.6%). After parthenogenetic activation of oocytes by ethanol/cycloheximide, cleavage stages were reached with rates of 51.9% (46 h IVM), 42.0% (12 h ageing) and 40.3% (26 h ageing), respectively. Furthermore, a significant higher proportion of long-term aged oocytes (26 h) showed pronuclear formation (8.6%) and fragmentation (7.9%) compared to non-aged oocytes (each 1.9%). It is concluded that both MAPK phosphorylation and cleavage rate after parthenogenetic activation decreased before alterations of nuclear stages could be detected during in vitro ageing of M II oocytes. A premature MAPK dephosphorylation of M II oocytes caused early anaphase II stages, but cleaved stages could not be achieved.

Down modulation of fibronectin messenger RNA in metastasizing rat prostatic cancer cells revealed by differential hybridization analysis.

To identify genes whose expression is down modulated in the process of metastasis, gene expression was analyzed in cell lines derived from Dunning R-3327 rat prostatic tumor sublines. A complementary DNA (cDNA) library from the anaplastic nonmetastasizing subline AT-1 was used for a differential hybridization analysis, using probes derived from mRNAs of the AT-1 and the metastasizing MAT-LyLu subline. In this way 14 cDNA clones were isolated representing 6 differentially expressed genes. The expression levels in a panel of tumor sublines measured with these cDNA clones were tested for correlation with the anaplastic non-metastasizing phenotype. One cDNA clone, designated pSE-1, whose expression was high in all tested sublines with that phenotype, appeared to represent the gene for fibronectin. To further investigate the down modulation of this gene, we studied its expression in AT-2 (anaplastic, nonmetastasizing tumor) and lines derived therefrom that exhibited a high metastatic potential after transfection with the v-Ha-ras oncogene. In the genetically manipulated metastasizing tumor sublines, fibronectin mRNA levels were approximately 4- to 8-fold lowered compared to the nonmetastasizing parental AT-2 line.

Human chromosome 21 determines growth factor dependence in human/mouse B-cell hybridomas.

Interleukin 6 (IL-6) serves as a growth factor for mouse plasmacytomas. As a model for IL-6-mediated growth of plasmacytomas, we study IL-6-dependent B-cell hybridomas, which can be generated through fusion of B lymphocytes with a plasmacytoma cell line, e.g., SP2/0. In the present report, we have investigated the peculiar behavior of B-cell hybridomas with respect to IL-6 dependence. We demonstrate that although newly generated hybridomas are IL-6 dependent, many hybridomas lose this dependency at frequencies as high as 50%, shortly after fusion. We speculated that the loss of IL-6-dependent growth is due to the well-known chromosomal instability of B-cell hybridomas. Consequently, loss of IL-6 dependence is the result of loss of a specific chromosome(s). This model implies the existence of an "IL-6 dependency" gene, the loss of which makes hybridomas capable of proliferating in the absence of IL-6. Because SP2/0 is IL-6 independent, the IL-6-dependent phenotype of B-cell hybridomas, and hence the IL-6 dependency gene, must be derived from the B lymphocyte. We have tested this model by generating human/mouse B-cell hybridomas through fusion of human B lymphocytes with SP2/0. We then analyzed the human chromosome content of 10 IL-6-dependent and 14 IL-6-independent subclones. From that analysis we concluded that the presence of human chromosome 21 correlated with IL-6 dependence. This correlation was confirmed by microcell fusion experiments in which a single copy of chromosome 21 was introduced into IL-6-independent hybridomas, resulting in reconstitution of the IL-6-dependent phenotype. We therefore conclude that chromosome 21 carries an IL-6 dependency gene.

Variable association of HLA-A2 in men with early-onset Alzheimer disease.

In the present study, we attempted to replicate the finding of an increased frequency of HLA-A2 in men with early-onset (less than or equal to 60 years) Alzheimer disease (AD). HLA data obtained on 167 patients (including 19 men with early-onset AD) from three geographic regions (North Carolina, Great Britain, and Finland) failed to replicate the result. A recent prospective study from Oregon, however, confirmed the association. Studies demonstrating the association suggest its presence in sporadic rather than familial AD. These results indicate a variable HLA/AD association, with some factor such as geographic region or disease familiality contributing to this variability.

Leucine-58 in the putative 5th helical region of human interleukin (IL)-6 is important for activation of the IL-6 signal transducer, gp130.

A model of the tertiary structure of human IL-6, derived from the crystal-structure of granulocyte-colony stimulating factor, reveals a 5th helical region in the loop between the first and second alpha-helix. To investigate the importance of this region for biological activity of IL-6, residues Glu-52, Ser-53, Ser-54, Lys-55, Glu-56, Leu-58, and Glu-60 were individually replaced by alanine. IL-6.Leu-58Ala displayed a 5-fold reduced biological activity on the IL-6 responsive human cell lines XG-1 and A375. This reduction in bioactivity was shown to be due to a decreased capacity of the mutant protein to trigger IL-6 receptor-alpha-chain-dependent binding to the IL-6 signal transducer, gp130.

Surface activity properties of cysteine-substituted C-terminal melittin analogues.

In order to extend our knowledge of factors important in the surface activity of melittin, cysteine was substituted for lysine-21 and lysine-21/glutamine-25 in a pair of synthetic peptide analogues. The first of these changes resulted in only modest effects on secondary structure (determined in 50% trifluoroethanol), emulsification and surface tension properties. Introduction of a second cysteine greatly reduced both the rate of surface tension decay and the equilibrium surface tension attained, although secondary structure (determined in 50% trifluoroethanol) was only slightly affected by this modification. This latter peptide completely lacked emulsification and haemolytic properties and was found to oligomerise readily due to the formation of intermolecular, disulphide bridges. These results indicate that oligomerisation abolishes surface activity in melittin.

Sequence analysis of small subunit ribosomal RNA genes and its use for detection and identification of Leishmania parasites.

The sequence of the most variable part of the small subunit ribosomal RNA (SSU rRNA) gene, comprising 800 bases, was analysed for 9 Leishmania taxa and compared with those of Trypanosoma brucei, Trypanosoma cruzi and Crithidia fasciculata. Considerable differences were observed between the sequence of the Leishmania taxa on the one hand and those of Crithidia and Trypanosoma on the other. Amongst the Leishmania taxa only a few point mutations were found, all located within 2 sequence blocks in the central part of the SSU rRNA gene, which are unique for Kinetoplastida. These unique sequences were used for the development of kinetoplastid-specific probes and a Leishmania-specific PCR assay of high sensitivity (less than 10 parasites could be detected). Based on the observed point-mutations an identification of the Leishmania parasites, according to complex, could be achieved by direct sequencing, restriction fragment analysis or single-stranded conformation polymorphism of the PCR-generated fragments.

Phosphorylation pattern of the p90rsk and mitogen-activated protein kinase (MAPK) molecule: comparison of in vitro and in vivo matured porcine oocytes.

The overall objective was to elucidate the phosphorylation pattern and activity of the kinase p90rsk, a substrate of mitogen-activated protein kinase (MAPK), during in vitro and in vivo maturation of pig oocytes. Cumulus-oocyte complexes were collected from slaughtered pigs and matured in vitro (0, 22, 26, 30, 34, 46 h) with and without the MEK inhibitor U0126. For in vivo maturation, gilts were stimulated with equine chorionic gonadotrophin (eCG) (600-800 IU). Maturation was induced 72 h later with hCG (500 IU). Oocytes were obtained surgically (0, 22, 30 h). The samples were submitted to electrophoresis and protein blotting analysis. Enhanced chemiluminescence was used for visualization. In vitro matured oocytes were further submitted to a commercially available radioactive kinase assay to determine kinase activity. It was shown that oocytes, as well as cumulus cells, already possess a partially phosphorylated p90rsk at the time of removal from follicles, with a further phosphorylation of the molecule occurring between 22-24 h after the initiation of culture, and in vivo maturation. The phosphorylation of p90rsk coincides with the phosphorylation of MAPK and can be prevented by U0126, indicating a MAPK-dependent phosphorylation of p90rsk. Phosphorylation of the in vivo matured oocytes occurred shown as a band of less than 200 kDa. This is presumably a molecule complex, with MAPK not being a component. Therefore, the p90rsk molecule in vivo exists as a dimer. Determination of kinase activity demonstrated decreasing enzyme activities. This led to the conclusion that the assay is not specific for p90rsk, instead measuring p70S6 kinase activities.

Mitogen-activated protein kinase phosphorylation patterns in pig oocytes and cumulus cells during gonadotrophin-induced resumption of meiosis in vitro.

The present study investigated the phosphorylation pattern of mitogen-activated protein kinase (MAPK) in cumulus-oocyte complexes (COCs) during spontaneous and FSH/LH-induced in vitro maturation (IVM). Both isoforms of MAPK were unphosphorylated in oocytes recovered immediately after liberation from follicles and became phosphorylated following 25 h incubation, corresponding to the time of germinal vesicle breakdown (GVBD). In contrast, MAPK was already phosphorylated in minimal amounts in cumulus cells at the time of liberation from follicles and phosphorylation of MAPK increased after 0.5 h incubation. Supplementation of medium with gonadotrophins intensified phosphorylation at 0.5 h incubation, demonstrating the early and rapid action of FSH/LH on MAPK phosphorylation. Phosphorylation of MAPK in cumulus cells peaked after 21 h of incubation, whereas MAPK was almost completely dephosphorylated at the end of incubation (45 h). During subsequent incubation in the absence of added gonadotrophins, between 5 and 10 h exposure to FSH/LH-supplemented medium was required to induce resumption of meiosis in COCs. Phosphorylation of MAPK in oocytes was prevented by the MEK inhibitor U0126, but the inhibitor reduced phosphorylation of MAPK in cumulus cells only during the first 2 h of IVM. The data support the hypothesis that two different MAPK phosphorylation events occurred following gonadotrophin stimulation, one in cumulus cells and the other in oocytes. In cumulus cells, FSH/LH induced early and rapid U0126-insensitive phosphorylation of MAPK, whereas U0126-susceptible MAPK phosphorylation took place in the oocyte itself around the time of GVBD.

A diploid-triploid (60,XX/90,XXY) intersex in a Holstein heifer.

The diploid/triploid (60,XX/90,XXY) condition in Bos taurus is very rare and only three cases have been published previously. The present animal exhibited an aplastic vulva, penis and clitoris agenesis, a male-like urethra located in a pseudoprepuce opening between the mammary complexes and a well developed M. rectipeninus. A normal (60,XX) female karyotype was detected in lymphocyte cultures whereas uterus and tendon cells revealed a 60,XX/90,XXY mixoploidy. Quantification of X and Y chromosome-specific sequences using RT-PCR revealed extraordinary high Y chromosome equivalents in the sample recovered from the male-like transformed vestibulum vaginae suggesting a causative relationship. The pathogenesis of the missing clitoris and penis, which is contrasted by the concomitant presence of a well developed M. rectipeninus, remains difficult to explain. A chimeric origin is suggested despite the fact that microsatellite analysis of the animal's blood cells displayed no un- usual allele accumulation.

The CD20/alphaCD20 'suicide' system: novel vectors with improved safety and expression profiles and efficient elimination of CD20-transgenic T cells.

Adoptive transfer of T lymphocytes is an attractive strategy for many experimental treatment strategies for cancer. Unfortunately, manipulated T cells could be responsible for serious adverse events. Retroviral CD20-transduced T cells may be able to control these unwanted effects. CD20-positive cells are sensitive to rituximab (RTX), a monoclonal antibody specific for CD20. This permits their selective elimination in vivo in case of adverse events. To this end, a system is required that permits efficient and safe transduction of donor T cells and effective elimination of CD20-positive T cells. We constructed different CD20-encoding retroviral vectors and investigated the impact of inclusion of the woodchuck post-transcriptional regulatory element (WPRE) and the chicken hypersensitivity site 4 insulator elements on the levels, homogeneity and stability of CD20 expression. Importantly, inclusion of either WPRE or insulator elements in the retroviral vector resulted in a dramatic improvement in the stability of CD20 expression. The insulator element also led to a much more homogeneous level of CD20 expression. We also show the efficient elimination of the CD20-transgenic T cells via RTX by different effector mechanisms. In conclusion, we have constructed CD20-encoding retroviral vectors with improved efficiency and safety profiles, which can be used as a suicide strategy.

T cell receptor-transgenic primary T cells as a tool for discovery of leukaemia-associated antigens.

Identification of a broad array of leukaemia-associated antigens is a crucial step towards immunotherapy of haematological malignancies. However, it is frequently hampered by the decrease of proliferative potential and functional activity of T cell clones used for screening procedures. Transfer of the genes encoding the T cell receptor (TCR) alpha and beta chains of leukaemia-specific clones into primary T cells may help to circumvent this obstacle. In this study, transfer of two minor histocompatibility antigen (minor H antigen)-specific TCRs was performed and the feasibility of the use of TCR-transgenic T cells for identification of minor H antigens through cDNA library screening was investigated. We found that TCR-transgenic cells acquired the specificity of the original clones and matched their sensitivity. Moreover, the higher scale of cytokine-production by TCR-transgenic T cells permits the detection of either small amounts of antigen-positive cells or cells expressing low amounts of an antigen. When applied in equal numbers, TCR-transgenic T cells and the original T cell clones produced similar results in the screening of a cDNA library. However, the use of increased numbers of TCR-transgenic T cells allowed detection of minute amounts of antigen, barely discernible by the T cell clone. In conclusion, TCR-transfer generates a large amount of functional antigen-specific cells suitable for screening of cDNA expression libraries for identification of cognate antigens.

Structural, biochemical and functional aspects of sperm-oocyte interactions in pigs.

Polyspermic fertilization is still a major issue in porcine IVF systems. New information is available to characterize the zona pellucida (ZP) at different developmental stages by scanning electron microscopy (SEM) and by confocal microscopy to show the distribution of ZP glycoproteins. SEM images indicated no differences between in vivo and in vitro matured oocytes; however a change in the surface structure between immature and matured oocytes, as well as between mature oocytes and preimplantation embryos was obvious. In addition, spermatozoa were more tightly fixed in the ZP of in vivo produced compared to the ZP of in vitro produced embryos. The ZP undergoes biochemical changes during maturation prior to fertilization. The acidity of the ZP increases during maturation as indicated by a shift of 1.3 pl units for ZPB/ZPC and 0.8 pl units for ZPA in 2D gel electrophoresis, which is based on increasing sulfation of the oligosaccharides during maturation. Mass spectrometry in combination with in-gel deglycosylation allowed the mapping of new glycosylation sites. Functionality of the ZP also depends on its maturation status. Induction of the acrosome reaction was delayed when capacitated spermatozoa were exposed to immature oocytes.

B-cell expansion in the presence of the novel 293-CD40L-sCD40L cell line allows the generation of large numbers of efficient xenoantigen-free APC.

BACKGROUND: CD40-activated B lymphocytes have been used successfully as potent APC for the induction of T-cell responses. However, the 3T3-CD40L cell line, regularly used for engagement of CD40 on the B-cell surface, is a potential source of xenoantigens. This may affect the specificity of T cells stimulated with CD40-activated B cells, especially when generation of T-cell lines specific for endogenously processed Ag is desired. METHODS: To develop a system that allows efficient expansion of B cells in the absence of sources of xenoantigens, we created a human 293-CD40L-sCD40L cell line that produces soluble CD40L and expresses CD40L on the cell surface. B cells from patients with hematologic malignancies were expanded on the 293-CD40L-sCD40L cells and used for stimulation of either naive or in vivo primed donor T cells in three HLA-identical patient-donor combinations. RESULTS: The 293-CD40L-sCD40L cell line was able to stimulate B-cell growth with an efficiency superior to that of the commonly used 3T3-CD40L cell line. In all cases T-cell lines and, subsequently, T-cell clones were generated that showed reactivity against patient and not donor B cells, suggesting their specificity for minor histocompatibility antigens (mHAg). DISCUSSION: B cells activated with GMP grade 293-CD40L-sCD40L can be used in a variety of applications. In particular, they may be suitable for ex vivo stimulation of T cells prior to donor lymphocyte infusion (DLI), which may enhance its graft versus leukemia (GvL) effect.