Rhesus monkeys (RM) were inoculated intrabronchially with graded doses of Mycobacterium tuberculosis (MTB) strains Erdman and H37Rv in an effort to produce a model of asymptomatic tuberculosis infection. Erdman strain produced active disease within 7-11 weeks regardless of dose. Low doses of H37Rv resulted in asymptomatic infections; high doses produced active disease within 11 weeks. Over a 4-month period of post-inoculation study, MTB culture-filtrate protein (CFP)-stimulated bronchoalveolar lavage cells (BALC) and blood mononuclear cells (PBMC) from monkeys with active disease (30 cfu Erdman-inoculated) or asymptomatic infection (200 cfu H37Rv-inoculated) produced similar significant quantities of mRNA encoding for IFN-gamma or TNF-alpha, but insignificant quantities of IL-4 mRNA. Differences were observed in antigen-induced in vitro blastogenic responses and serum anti-lipoarabinomannan (LAM) antibody responses in animals with active compared with asymptomatic MTB infections. The results indicate that RM are a good model for the study of asymptomatic tuberculosis infections using low doses of H37Rv.
The oviduct epithelium undergoes marked morphological and functional changes during the estrous cycle. It has been shown that a dramatic change in the frequencies of ciliated and non-ciliated cells occurs during the estrous cycle. At estrus the epithelium consists of secretory and ciliated cells and at diestrus mainly of ciliated cells. The oviduct provides the microenvironment for sperm capacitation, fertilization, and early cleavage-stage embryonic development. At the molecular level, only a few genes or proteins are known that change during the estrous cycle and which may be important for fertility, so as the bovine oviduct-specific glycoprotein, the major secretory protein in the oviduct. Therefore, we studied systematically the changes in gene expression in bovine ipsilateral oviduct epithelial cells at estrus and diestrus. To identify differentially expressed genes, a combination of subtracted cDNA libraries and cDNA array hybridization was used. Two subtracted libraries were produced to enrich cDNAs of upregulated genes at estrus and at diestrus. A total of 1536 cDNA clones of each library were analyzed with radioactively (33-P) labeled probes generated from the oviduct epithelial cells of six Simmental heifers, three of them slaughtered at Day 0 (estrus) and three at Day 12 after standing heat (diestrus). After normalization of the raw data and statistical analysis, all cDNAs showing significant differences in their expression levels at estrus compared to diestrus were sequenced. Sequencing revealed 84 different cDNAs; 42 of them matched bovine genes or their human/mouse homologs with known functions, and 42 matched genes without a known function. Half of the genes (n = 42) were expressed at a higher level at estrus; for the other (n = 42) expression levels were higher at diestrus. The regulated genes or their products represented a variety of functional classes, such as genes of the secretory pathway, genes involved in transcription regulation, cell-surface proteins, cell-cell interaction proteins, secreted proteins, members of signal transduction pathways, immune-related proteins, and some enzymes. The identification of genes differentially regulated in ipsilateral oviduct epithelial cells at estrus v. diestrus is the first step of a systematic analysis of differential gene expression during the estrous cycle. Further studies will follow, focusing on different compartments of the bovine oviduct and additional times of the estrous cycle. EXPRESSION PATTERN OF CERTAIN DEVELOPMENTALLY IMPORTANT GENES IN BOVINE NUCLEAR TRANSFER EMBRYOS PRODUCED USING CELL LINES OF DIFFERENT EFFICIENCY Z. Beyhan and N.L. FirstDepartment of Animal Science, University of Wisconsin-Madison, WI, USA. email: zbeyhan@wisc.edu Developmental abnormalities associated with the cloning process suggest that reprogramming of donor nuclei into an embryonic state may not be fully completed in most of the cloned animals. One of the areas of interest in this respect is the analysis of gene expression patterns in nuclear transfer embryos to ...
226 Reproduction, Fertility and DevelopmentEmbryo Manipulation 0.035, respectively). In addition, at 4 weeks of age, the in vivo males became significantly lighter when compared to the naturally mated males (P = 0.034). At 8 weeks of age, the in vivo females had a significantly elevated systolic blood pressure when compared to the in vitro females (P = 0.003); however, at 21 weeks of age, both in vitro males and females had a significantly elevated blood pressure when compared to in vivo offspring (P < 0.003). At 8, 15, and 21 weeks of age, offspring derived from transferred embryos developed with significantly elevated systolic blood pressure when compared to non-embryo transfer offspring (P < 0.05). No significant differences in serum angiotensin-converting enzyme activity (a potent regulator of systolic blood pressure) was observed between the treatment groups. Significantly altered liver:body weight ratios were observed between the in vitro and in vivo males, and between the in vitro and the naturally mated (6) females (P < 0.038). All of the above data are independent of litter size. These data support the hypothesis that early embryo environment can influence postnatal growth and cardiovascular physiology. The present study was conducted to determine the effects of hexoses on in vitro maturation, fertilization, and development of porcine oocytes. In the first experiment, porcine oocytes were matured in modified North Carolina State University (NCSU)-37 medium supplemented with 0.6 mM cysteine, 1 mM dibutyryl cyclic AMP (dbc AMP), 10 IU/mL equine chorionic gonadotrophin, 10 IU/mL human chorionic gonadotrophin, 50 µ/mL gentamycin (Sigma-Aldrich, Tokyo, Japan), 10% (v/v) porcine follicular fluid, and various hexoses (glucose, fructose, and galactose) at various concentrations of 0 (control), 2.5, 5.5, and 10 mM. They were subsequently cultured in the maturation medium without hormones and dbcAMP for an additional 22 h. Fertilization was performed according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033-1041; 15 oocytes were co-incubated with 1 million frozen thawed sperm/mL in fertilization medium for 5 h. Supplementation of either glucose (2.5 or 5.5 mM) or fructose (5.5 mM) in the maturation medium significantly increased the percentages of maturation to metaphase II (68.5%, 79.4%, and 70.2%, respectively) and monospermic fertilization of oocytes (55.0%, 64.5%, and 58.9%, respectively), as compared with control group (metaphase II: 52.8%; monospermic: 42.7%; P < 0.05). Supplementation of galactose had no effect on the meiotic maturation and monospermic fertilization of oocytes. In the second experiment, presumptive zygotes were cultured in modified NCSU-37 supplemented with 4 mg/mL BSA, 0.17 mM sodium pyruvate, 2.73 mM sodium lactate, and 50 µg/mL gentamycin. The cleaved embryos were collected at Day 3 after in vitro fertilization and then cultured for a further 4 days in modified NCSU-37 medium supplemented with 5.5 mM of glucose, fructose, or galactose. The percentages of blastocyst formation, calculated ...
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