Effect of 17beta-estradiol on the in vitro maturation of bovine oocytes

Although 1 microg/ml of 17beta-estradiol (E2) is often used in routine in vitro maturation (IVM) and in vitro fertilization (IVF), its effect remains controversial. The objective of our study was to investigate the effects of E2 on bovine oocyte IVM and subsequent embryo development, using a defined medium. Bovine cumulus oocyte complexes (COCs), aspirated from 2 to 8 mm follicles of slaughterhouse ovaries, were matured in TCM199 in the presence of 1 microg/ml E2 with or without 0.05 IU/ml recombinant hFSH. Cultures without E2, FSH or both served as controls. COCs were matured for 22 h at 39 degrees C in a humidified atmosphere of 5% CO2 in air. To investigate the effect of E2 with and without FSH on nuclear maturation, COCs were fixed after maturation and the nuclear stage was assessed following DAPI staining. Similarly, denuded oocytes (DO) were matured in the presence of E2 and the nuclear stage assessed after 22 h. To investigate the effect of E2 with and without FSH during IVM on subsequent embryo development, in vitro matured COCs were fertilized in vitro and after removal of the cumulus cells, the presumed zygotes were cocultured on BRL monolayer for 11 days. At Day 4, the number of cleaved embryos, and at Days 9 and 11, the number of blastocysts, were assessed. Addition of 1 microg/ml E2 to TCM199 significantly decreased the percentage of Metaphase II (MII) compared to control (56.3 and 74.0%, respectively), and increased the percentage of nuclear aberrations compared to control (13.3 and 2.1%, respectively). The negative effect of E2 on nuclear maturation was stronger when DO were matured; 25.1 and 60.0% of the oocytes reached MII stage for the E2 and control groups, respectively. When COCs were matured in TCM199 supplemented with FSH, the addition of 1 microg/ml E2 did not influence the proportion of MII oocytes, although a higher percentage of nuclear aberrations as compared to control was observed. Presence of E2 during IVM also decreased the blastocyst rate (14.4 and 10.0% for control and E2 groups, respectively). However, when FSH was present, the addition of E2 had no effect on the cleavage rate and blastocyst formation (20.3 and 21.7% for control and E2 groups, respectively). In conclusion, supplementation of 1 microg/ml E2 to a serum free maturation medium negatively affects bovine oocyte nuclear maturation and subsequent embryo development. Although these effects are attenuated in the presence of FSH, we strongly suggest omission of E2 in routine maturation protocols of bovine oocytes.     

Regiospecific oxygenation of alkenones in the benthic haptophyte Chrysotila lamellosa Anand HAP 17

Two groups of previously unidentified C37-C39 epoxyalkenones and alkenediones were detected in late stationary phase cultures of the haptophyte microalga Chrysotila lamellosa. The formation of these compounds is attributed to the involvement of enzymatic processes acting specifically on the C-21 or C-22 allylic carbon and the omega15 double bond of methyl and ethyl alkenones respectively. Thus, the epoxyalkenones appear to be derivatives of alkenones where the omega15 double bond is oxidized to the epoxide. These epoxyalkenones disappear as the cells age to be replaced by a series of alkenediones. The structures of these compounds indicate that they are derivatives of methyl and ethyl alkenones with an additional carbonyl group on the C-21 or C-22 carbon respectively and without the omega15 double bond. We propose that these compounds are formed by an initial regiospecific lipoxygenase-catalyzed peroxidation of methyl and ethyl alkenones on their C-21 or C-22 allylic carbon, respectively. Lipohydroperoxidase-catalyzed homolytic cleavage of the O-O bond could then result in the formation of conjugated ketones which may then undergo a saturation reaction to form the diketones identified. This work demonstrates that alkenones can be degraded by enzymatic reactions in senescent cells, and by implication this could also occur in the natural environment.     

Phylogenetic analysis using insertion sequence fingerprinting in Escherichia coli

Chromosomal DNA from 23 closely related, pathogenic strains of Escherichia coli was digested and probed for the insertion sequences IS1, IS2, IS4, IS5, and IS30. Under the assumption that elements residing in DNA restriction fragments of the same apparent length are identical by descent, parsimony analysis of these characters yielded a unique phylogenetic tree. This analysis not only distinguished among bacterial strains that were otherwise identical in their biochemical characteristics and enzyme electrophoretic mobilities, but certain aspects of the topology of the tree were consistent across several unrelated insertion elements. The distribution of IS elements was then reexamined in light of the inferred phylogenetic relationships to investigate the biological properties of the elements, such as rates of insertion and deletion, and to discover apparent recombinational events. The analysis shows that the pattern of distribution of insertion elements in the bacterial genome is sufficiently stable for epidemiological studies. Although the rate of recombination by conjugation has been postulated to be low, at least two such events appear to have taken place.      

Sequence, organization, and evolution of the A+T region of Drosophila melanogaster mitochondrial DNA

The long (4.6-kb) A+T region of Drosophila melanogaster mitochondrial DNA has been cloned and sequenced. The A+T region is organized in two large arrays of tandemly repeated DNA sequence elements, with nonrepetitive intervening and flanking sequences comprising only 22% of its length. The first repeat array consists of five repeats of 338-373 bp. The second consists of four intact 464-bp repeats and a fifth partial repeat of 137 bp. Three DNA sequence elements are found to be highly conserved in D. melanogaster and in several Drosophila species with short A+T regions. These include a 300-bp DNA sequence element that overlaps the DNA replication origin and two thymidylate stretches identified on opposite DNA strands. We conclude that the length heterogeneity observed in the A+T regulatory region in mitochondrial DNAs from the genus Drosophila results from the expansion (and contraction) of the number of repeated DNA sequence elements. We also propose that the 300-bp conserved DNA sequence element, in conjunction with another primary sequence determinant, perhaps the adjacent thymidylate stretch, functions in the regulation of mitochondrial DNA replication.      

Microbial Transport, Survival, and Succession in a Sequence of Buried Sediments

Abstract Two chronosequences of unsaturated, buried loess sediments, ranging in age from <10,000 years to >1 million years, were investigated to reconstruct patterns of microbial ecological succession that have occurred since sediment burial. The relative importance of microbial transport and survival to succession was inferred from sediment ages, porewater ages, patterns of abundance (measured by direct counts, counts of culturable cells, and total phospholipid fatty acids), activities (measured by radiotracer and enzyme assays), and community composition (measured by phospholipid fatty acid patterns and Biolog substrate usage). Core samples were collected at two sites 40 km apart in the Palouse region of eastern Washington State, near the towns of Washtucna and Winona. The Washtucna site was flooded multiple times during the Pleistocene by glacial outburst floods; the Winona site elevation is above flood stage. Sediments at the Washtucna site were collected from near surface to 14.9 m depth, where the sediment age was approximately 250 ka and the porewater age was 3700 years; sample intervals at the Winona site ranged from near surface to 38 m (sediment age: approximately 1 Ma; porewater age: 1200 years). Microbial abundance and activities declined with depth at both sites; however, even the deepest, oldest sediments showed evidence of viable microorganisms. Same-age sediments had equal quantities of microorganisms, but different community types. Differences in community makeup between the two sites can be attributed to differences in groundwater recharge and paleoflooding. Estimates of the microbial community age can be constrained by porewater and sediment ages. In the shallower sediments (<9 m at Washtucna, <12 m at Winona), the microbial communities are likely similar in age to the groundwater; thus, microbial succession has been influenced by recent transport of microorganisms from the surface. In the deeper sediments, the populations may be considerably older than the porewater ages, since microbial transport is severely restricted in unsaturated sediments. This is particularly true at the Winona site, which was never flooded.     

Tissue type-specific expression of intermediate filament proteins in a cultured epithelial cell line from bovine mammary gland

Different clonal cell lines have been isolated from cultures of mammary gland epithelium of lactating cow’s udder and have been grown in culture media containing high concentrations of hydrocortisone, insulin, and prolactin. These cell (BMGE+H), which grow in monolayers of typical epithelial appearance, are not tightly packed, but leave intercellular spaces spanned by desmosomal bridges. The cells contain extended arrays of cytokeratin fibrils, arranged in bundles attached to desmosomes. Gel electophoresis show that they synthesize cytokeratins similar, if not identical, to those found in bovine epidermis and udder, including two large (mol wt 58,500 and 59,000) and basic (pH range: 7-8) and two small (mol wt 45,500 and 50,000) and acidic (pH 5.32 and 5.36) components that also occur in phosphorylated forms. Two further cytokeratins of mol wts 44,000 (approximately pH 5.7) and 53,000 (pH 6.3) are detected as minor cytokeratins in some cell clones. BMGE+H cells do not produce vimentin filaments as determined by immunofluorescence microscopy and gel electrophoresis. By contrast, BMGE-H cells, which have emerged from the same original culture but have been grown without hormones added, are not only morphologically different, but also contain vimentin filaments and a different set of cytokeratins, the most striking difference being the absence of the two acidic cytokeratins of mol wt 50,000 and 45,500. Cells of the BMGE+H line are characterized by an unusual epithelial morphology and represent the first example of a nonmalignant permanent cell line in vitro that produces cytokeratin but not vimentin filaments. The results show that (a) tissue-specific patterns of intermediate filament expression can be maintained in permanent epithelial cell lines in culture, at least under certain growth conditions; (b) loss of expression of relatively large, basic cytokeratins is not an inevitable consequence of growth of epithelial cells in vitro. Our results further show that, during culturing, different cell clones with different cytoskeletal composition can emerge from the same cell population and suggest that the presence of certain hormones may have an influence on the expression of intermediate filament proteins.     

Selective neutrality of glucose-6-phosphate dehydrogenase allozymes in Escherichia coli

Six naturally occurring alleles representing four electromorphs of the enzyme glucose-6-phosphate dehydrogenase were transferred by P1- mediated transduction from natural isolates of Escherichia coli into the genetic background of E. coli K12 and were studied in pairwise competition in chemostats limited for glucose in order to estimate differences in growth rate associated with the alleles. Although the level of resolution of such experiments is a growth rate differential of approximately 0.002 h-1, no significant differences among the strains were found. Studies of apparent Km and Vmax in crude enzyme extracts of the strains also failed to reveal any significant differences among the electromorphs. These results support the view that the alleles are selectively neutral or nearly neutral under these conditions.