Laparoscopical intrauterine insemination with different doses of fresh,conserved, and frozen semen for the production of ovine zygotes

The objective of the present study was to increase the efficiency in the production of ovine zygotes suitable for microinjection via laparoscopical intrauterine insemination. In the first part of the study, 71 ewes of three different breeds were inseminated with one of two different insemination doses (50 x 10(6) or 300 x 10(6) sperm per inseminate) and semen was either freshly diluted, liquid conserved, or frozen/thawed, or females were mated by a fertile ram (controls). In the second part, a total of 46 ewes was inseminated with 300 x 10(6) freshly diluted sperm to verify the findings from part 1 and to unravel effects of breed and age of donor ewe. The oviducts were flushed 24-26 h after insemination and the success of insemination was assessed by microscopical examination. Recovery rates were 78.0+/-26.4 and 72.1+/-24.6% in parts 1 and 2 of the study, respectively. Of these oocytes 62.3 and 62.8% (parts 1 and 2, respectively) were fertilized. In part 1, the highest proportion (64.7%) of pronuclear stages was observed in the group inseminated with 300 x 10(6) freshly diluted semen and was significantly higher compared to the groups inseminated with 50 x 10(6) freshly diluted semen (25.5%, P<0.001), 300 x 10(6) liquid conserved semen (49.0%, P<0.001), or 50 x 10(6) frozen/thawed semen (39.6%, P<0.05). In the control group, the proportion of pronuclear stages amounted to 60.2%. Irrespective of the type of sperm conservation, the overall fertilization rate (zygotes plus 2-cell stages) was higher (P<0.05) following insemination with 300 x 10(6) sperm (68.2%) compared to 50 x 10(6) sperm (56.8%). In part 2, the proportion of pronuclear stages reached 54.2% with an overall fertilization rate of 62.9%. These results were affected by breed and age of the donor as crossbred and younger (<3 years) animals yielded the highest proportion of pronuclear stages. The present study shows that freshly diluted semen at a dosage of 300 x 10(6) sperm yields the highest fertilization rates, the greatest proportion of pronuclear stages and the lowest proportion of mature unfertilized oocytes. Further increases in yields of pronuclear stages can possibly be achieved by selection of sheep from the best suited breed and younger than 3 years of age.     

Development of a Listeria monocytogenes EGDe partial proteome reference map and comparison with the protein profiles of food isolates

A partially annotated proteome reference map of the food pathogen Listeria monocytogenes was developed for exponentially growing cells under standardized, optimal conditions by using the sequenced strain EGDe (serotype 1/2a) as a model organism. The map was developed by using a reproducible total protein extraction and two-dimensional (2-D) polyacrylamide gel electrophoresis analysis procedure, and it contained 33 identified proteins representing the four main protein functional classes. In order to facilitate analysis of membrane proteins, a protein compartmentalization procedure was assessed. The method used provided partial fractionation of membrane and cytosolic proteins. The total protein 2-D profiles of three serotype 1/2a strains and one serotype 1/2b strain isolated from food were compared to the L. monocytogenes EGDe proteome. An average of 13% of the major protein spots in the food strain proteomes were not matched in the strain EGDe proteome. The variation was greater for the less intense spots, and on average 28% of these spots were not matched. Two of the proteins identified in L. monocytogenes EGDe were missing in one or more of the food isolates. These two proteins were proteins involved in the main glycolytic pathway and in metabolism of coenzymes and prosthetic groups. The two corresponding genes were found by PCR amplification to be present in the four food isolates. Our results show that the L. monocytogenes EGDe reference map is a valuable starting point for analyses of strains having various origins and could be useful for analyzing the proteomes of different isolates of this pathogen.     

Selective removal of the selenocysteine tRNA [Ser]Sec gene (Trsp) in mouse mammary epithelium

Mice homozygous for an allele encoding the selenocysteine (Sec) tRNA [Ser]Sec gene (Trsp) flanked by loxP sites were generated. Cre recombinase-dependent removal of Trsp in these mice was lethal to embryos. To investigate the role of Trsp in mouse mammary epithelium, we deleted this gene by using transgenic mice carrying the Cre recombinase gene under control of the mouse mammary tumor virus (MMTV) long terminal repeat or the whey acidic protein promoter. While both promoters target Cre gene expression to mammary epithelium, MMTV-Cre is also expressed in spleen and skin. Sec tRNA [Ser]Sec amounts were reduced by more than 70% in mammary tissue with either transgene, while in skin and spleen, levels were reduced only with MMTV-Cre. The selenoprotein population was selectively affected with MMTV-Cre in breast and skin but not in the control tissue, kidney. Moreover, within affected tissues, expression of specific selenoproteins was regulated differently and often in a contrasting manner, with levels of Sep15 and the glutathione peroxidases GPx1 and GPx4 being substantially reduced. Expression of the tumor suppressor genes BRCA1 and p53 was also altered in a contrasting manner in MMTV-Cre mice, suggesting greater susceptibility to cancer and/or increased cell apoptosis. Thus, the conditional Trsp knockout mouse allows tissue-specific manipulation of Sec tRNA and selenoprotein expression, suggesting that this approach will provide a useful tool for studying the role of selenoproteins in health.     

Impaired differentiation and lactational failure of Erbb4-deficient mammary glands identify ERBB4 as an obligate mediator of STAT5

The ERBB family of type 1 receptor tyrosine kinases and their ligands have crucial functions during mammopoiesis, but the signaling networks that ultimately regulate ERBB activity in the breast have remained elusive. Here, we show that mice with Cre-lox mediated deletions of both Erbb4 alleles within the developing mammary gland (Erbb4(Flox/Flox)Wap-Cre) fail to accumulate lobuloalveoli or successfully engage lactation at parturition owing, in part, to impaired epithelial proliferation. Analysis of the mammary differentiation factor STAT5 by immunohistochemistry and western blot revealed a complete ablation of STAT5 activation in Erbb4(Flox/Flox)Wap-Cre mammary epithelium at parturition. Consistent with disrupted STAT5 function, Erbb4(Flox/Flox)Wap-Cre mammary glands at parturition failed to express the mammary epithelial differentiation marker NPT2B. Defects in epithelial functional differentiation at parturition were accompanied by a profound reduction in expression of the STAT5-regulated milk genes casein beta and whey acidic protein. We propose that ERBB4 functions as an essential mediator of STAT5 signaling, and that loss of STAT5 activity contributes to the impaired functional differentiation of mammary glands observed in mice containing conditional Erbb4 deletions.     

Characterisation of estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity in the human brain

Estrogens play a crucial role in multiple functions of the brain and the proper balance of inactive estrone and active estradiol-17beta might be very important for their cerebral effects. The interconversion of estrone and estradiol-17beta in target tissues is known to be catalysed by a number of human 17beta-hydroxysteroid dehydrogenase (17beta-HSD) isoforms. The present study shows that enzyme catalysed interconversion of estrone and estradiol-17beta occurs in the human temporal lobe. The oxidative cerebral pathway preferred estradiol-17beta to Delta(5)-androstenediol and testosterone, whereas the reductive pathway preferred dehydroepiandrosterone (DHEA) to Delta(4)-androstenedione and estrone. An allosteric Hill kinetic for NAD-dependent oxidation of estradiol-17beta was observed, whereas a typical Michaelis-Menten kinetic was shown for NADPH-dependent reduction of estrone. Investigations of the interconversion of estrogens in cerebral neocortex (CX) and subcortical white matter (SC) preparations of brain tissue from 12 women and 10 men revealed no sex-differences, but provide striking evidence for the presence of at least one oxidative membrane-associated 17beta-HSD and one cytosolic enzyme that catalyses both the reductive and the oxidative pathway. Membrane-associated oxidation of estradiol-17beta was shown to be significantly higher in CX than in SC (P<0.05), whereas the cytosolic enzyme activities were significantly higher in SC than in CX (P<0.0005). Finally, real-time RT-PCR analyses revealed that besides 17beta-HSD types 4 and 5 also the isozymes type 7, 8, 10 and 11 show substantial expression in the human temporal lobe. The characteristics of the isozymes lead us to the conclusion that cytosolic 17beta-HSD type 5 is the best candidate for the observed cytosolic enzyme activities, whereas the data gave no clear answer to the question, which enzyme is responsible for the membrane-associated oxidation of estradiol-17beta. In conclusion, the study strongly suggests that different cell types and different isozymes are involved in the cerebral interconversion of estrogens, which might play a pivotal role in maintaining the functions of the central nervous system.     

Micromechanics and anatomical changes during early ontogeny of two lianescent Aristolochia species

 The mechanical properties of young stems of Aristolochia macrophylla Lam. and Aristolochia brasiliensis Mart. et Zucc. were studied during elongation growth and primary differentiation. Data for the modulus of elasticity, for the viscoelastic behaviour caused by longitudinal tension and for the shear modulus resulting from torsion around a longitudinal axis were related to the underlying structural changes by quantitative analysis of stem anatomy, tissue distribution, ultrastructure, and cell wall biochemistry. The orientation of cellulose microfibrils was determined by light microscopy and small-angle X-ray diffraction, and the lignin content was determined by thioglycolic acid derivatization and spectroscopic quantification. It was demonstrated that the increase in stability during early development is due to the complementary effects of increase in cell wall material, lignification, and cellulose microfibril alignment. A detailed micromechanical model, considering internal prestresses, is proposed to explain the characteristic biphasic stress-strain behaviour as well as the strain-hardening observed. Received: 22 March 1999 / Accepted 9 September 1999     

Transgenic Arabidopsis plants can accumulate polyhydroxybutyrate to up to 4% of their fresh weight

 Transgenic Arabidopsis thaliana (L.) Heynh. plants expressing the three enzymes encoding the biosynthetic route to polyhydroxybutyrate (PHB) are described. These plants accumulated more than 4% of their fresh weight (≈40% of their dry weight) in the form of PHB in leaf chloroplasts. These very high producers were obtained and identified following a novel strategy consisting of a rapid GC-MS analysis of a large number of transgenic Arabidopsis plants generated using a triple construct, thus allowing the parallel transfer of all three genes necessary for PHB synthesis in a single transformation event. The level of PHB produced was 4-fold greater than previously published values, thus demonstrating the large potential of plants to produce this renewable resource. However, the high levels of the polymer produced had severe effects on both plant development and metabolism. Stunted growth and a loss of fertility were observed in the high-producing lines. Analysis of the metabolite composition of these lines using a GC-MS method that we have newly developed showed that the accumulation of high levels of PHB was not accompanied by an appreciable change in either the composition or the amount of fatty acids. Substantial changes were, however, observed in the levels of various organic acids, amino acids, sugars and sugar alcohols. Received: 2 February 2000 / Accepted: 31 March 2000     

Adjusting for Ordinal Covariates by Inducing a Partial Ordering

In the context of randomized clinical trials, multiplicity arises in many forms. One prominent example is when a key endpoint is measured and analyzed both at baseline and after treatment. It is common to analyze each separately, but more efficient to adjust the post‐treatment comparisons for the baseline values. Adjustment techniques generally treat the covariate (baseline value, in this case) as either nominal or continuous. Either is problematic when applied to an ordinal covariate, the former because it fails to exploit the natural ordering and the latter because it relies on an artifical notion of linear prediction and differences between values. We propose new methods for adjusting for ordinal covariates without having to treat them as nominal or continuous. Specifically, the information‐preserving composite endpoint consists of the pair of values for each patient, one at baseline and one after treatment. Some of these patterns will indicate more improvement than others, yet some pairs of patterns are not comparable. Hence, the ordering is only partial. We develop an approach to testing and deriving estimators of magnitudes of the treatment effect based on comparing each observation in one group to each observation in the other group to which it is comparable. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Historical Invasion Records Can Be Misleading: Genetic Evidence for Multiple Introductions of Invasive Raccoons (Procyon lotor) in Germany

Biological invasions provide excellent study systems to understand evolutionary, genetic and ecological processes during range expansions. There is strong evidence for positive effects of high propagule pressure and the associated higher genetic diversity on invasion success, but some species have become invasive despite small founder numbers. The raccoon (Procyon lotor) is often considered as a typical example for such a successful invasion resulting from a small number of founders. The species’ largest non-native population in Germany is commonly assumed to stem from a small number of founders and two separate founding events in the 1930s and 1940s. In the present study we analyzed 407 raccoons at 20 microsatellite loci sampled from the invasive range in Western Europe to test if these assumptions are correct. Contrary to the expectations, different genetic clustering methods detected evidence for at least four independent introduction events that gave rise to genetically differentiated subpopulations. Further smaller clusters were either artifacts or resulted from founder events at the range margin and recent release of captive individuals. We also found genetic evidence for on-going introductions of individuals. Furthermore a novel randomization process was used to determine the potential range of founder population size that would suffice to capture all the alleles present in a cluster. Our results falsify the assumption that this species has become widespread and abundant despite being genetically depauperate and show that historical records of species introductions may be misleading.