Laser-assisted zona pellucida thinning does not facilitate hatching and may disrupt the in vitro hatching process: a morphokinetic study in the mouse

STUDY QUESTION: Does laser-assisted zona thinning of cleavage stage mouse embryos facilitate hatching in vitro? SUMMARY ANSWER: No, unlike laser zona opening, zona thinning does not facilitate embryo hatching. WHAT IS KNOWN ALREADY: Artificial opening of the zona pellucida facilitates hatching of mouse and human embryos. Laser-assisted zona thinning has also been used for the purpose of assisted hatching of human embryos but it has not been properly investigated in an animal model; thinning methods have produced inconsistent clinical results. STUDY DESIGN, SIZE, DURATION: Time-lapse microscopy was used to study the hatching process in the mouse after zona opening and zona thinning; a control group of embryos was not zona-manipulated but exposed to the same laser energy. PARTICIPANTS/MATERIALS, SETTING, METHODS: Eight-cell CB6F1/J mouse embryos were pooled and allocated to three groups (n = 56 per group): A control group of embryos that were exposed to a dose of laser energy focused outside the zona pellucida (zona intact); one experimental group of embryos in which the zona pellucida was opened by complete ablation using the same total number of pulses as the control group; a second experimental group of embryos in which the zona pellucida was thinned to establish a smooth lased area using the same number of pulses as used in the other two groups. The width of the zona opening was 25 mum and width of the thinned area was 35 mum. Development was monitored by time-lapse microscopy. Overall treatment differences for continuous variables were analyzed by analysis of variance and pairwise comparisons using the Student t-test allowing for unequal variances, while for categorical data, a standard chi-squared test was utilized for all pairwise comparisons. MAIN RESULTS AND THE ROLE OF CHANCE: The frequency of complete hatching was 33.9% in the control group, 94.4% after zona opening, and 39.3% after zona thinning (overall group comparison, P \u3c 0.0001). Overall, 60.7% of the zona-thinned embryos did not complete the hatching process and remained trapped within the zona; when they did hatch, they did not necessarily hatch from the zona-thinned area. Hatching in about one-third of the zona-intact embryos began with breaches at multiple sites by small groups of cells. Likewise, 53.6% of zona-thinned embryos had multiple breaches, always involving an area outside the thinned zone. Zona opening decreased multiple breaching and led to blastocyst escape an average of 14 h earlier than zona-thinned embryos and 5.5 h before control embryos (P = 0.0003). LIMITATIONS, REASONS FOR CAUTION: The experiments presented here were limited to in vitro experiments performed in the mouse. Whether human embryos would behave the same way under similar circumstances is unknown. We postulate that zona thinning is not beneficial in human embryos. WIDER IMPLICATIONS OF THE FINDINGS: The experiments demonstrate that zona thinning is not equivalent to zona opening for assisted hatching. The study provides reason for systematic reviews of assisted hatching trials to take the method of assisted hatching into consideration and not combine the results of zona thinning and zona opening procedures. STUDY FUNDING/COMPETING INTERESTS: Institutional funds were used for the study. No competing interests are declared

Advanced atomic force microscopy techniques

Cataloged from PDF version of article.Although its conceptual approach is as simple as the technique used in record players already introduced in the 19th century, the invention of the atomic force microscope (AFM) in 1986 by Binnig, Quate, and Gerber was a milestone for nanotechnology. The scanning tunneling microscope (STM), introduced some years earlier, had already achieved atomic resolution, but is limited to conductive surfaces. Since its operational principle is based on the detection of the forces acting between tip and sample, this restriction does not exist for the AFM. Consequently, atomic force microscopy quickly became the standard tool for nanometer-scale imaging of all types of surfaces in all environments. True atomic resolution was first achieved in the 1990s. The most convincing results, however, were restricted to the so-called noncontact mode in vacuum for a long time, but recent technical developments overcame this limitation, and atomic-resolution imaging is now also a standard in liquids. Beyond pushing the resolution limit to the picometer range, the invention of the AFM triggered the development of a growing number of new scanning probe methods and approaches, ranging from an expansion of the properties that can be mapped to the active manipulation of surfaces and small particles. Practically every month, reports on the growing capabilities of AFMs appear. Nearly every physical effect that influences the tip–sample interaction has been used to improve existing modes and to develop new ones. For example, many recently presented techniques include the excitation of higher cantilever oscillation modes; it is amazing in how many ways the shaking of a simple cantilever can improve our knowledge about the tip–sample interaction. Another direction is high-speed atomic force microscopy, which is one of the eminent challenges that need to be solved in order to allow the in situ observation of biological processes. Data acquisition times have already reached the millisecond range, enabling the visualization of the dynamic behavior of biological molecules and cells. Other recent accomplishments include imaging of organic molecules with unprecedented resolution, full three-dimensional mapping of surface force fields, and the imaging and discrimination of individual chemical bonds. The development of advanced techniques is the focus of this Thematic Series, following the Thematic Series “Scanning probe microscopy and related techniques” edited by Ernst Meyer and the Thematic Series “Noncontact atomic force microscopy” edited by Udo Schwarz. The articles that are part of the series demonstrate that, despite its 25 years of history, the AFM is still far from reaching its limits, and today’s developments are far-reaching. As the number of research groups utilizing advanced atomic force microscopy techniques increases with each passing year, the technical improvements, data-acquisition approaches, analysis procedures, user friendliness, and application areas of the technique further diversify. With this Thematic Series, it is our intention to stimulate these improvements. We thank all authors for contributing their excellent work to this series. Furthermore, we acknowledge all referees for their promptly provided reports keeping the publication times short and attractive for contributors. Finally, we are grateful to the open access policy of the Beilstein Journal of Nanotechnology providing the ground for unrestricted discussions on advanced atomic force microscopy techniques. Thilo Glatzel, Hendrik Hölscher, Thomas Schimmel, Mehmet Z. Baykara, Udo D. Schwarz and Ricardo Garcia December 201

The Method-specific Certification of the Cholesterol and Triglyceride Contents of a Pure and an Adulterated Butter Fat Reference Material.

Abstract not availableJRC.D-Institute for Reference Materials and Measurements (Geel

Detection of genetic incompatibilities in non-model systems using simple genetic markers: hybrid breakdown in the haplodiploid spider mite Tetranychus evansi

When two related species interbreed, their hybrid offspring frequently suffer from reduced fitness. The genetics of hybrid incompatibility are described by the Bateson–Dobzhansky–Muller (BDM) model, where fitness is reduced by epistatic interactions between alleles of heterospecific origin. Unfortunately, most empirical evidence for the BDM model comes from a few well-studied model organisms, restricting our genetic understanding of hybrid incompatibilities to limited taxa. These systems are predominantly diploid and incompatibility is often complete, which complicates the detection of recessive allelic interactions and excludes the possibility to study viable or intermediate stages. Here, we advocate research into non-model organisms with haploid or haplodiploid reproductive systems and incomplete hybrid incompatibility because (1) dominance is absent in haploids and (2) incomplete incompatibility allows comparing affected with unaffected individuals. We describe a novel two-locus statistic specifying the frequency of individuals for which two alleles co-occur. This approach to studying BDM incompatibilities requires genotypic characterization of hybrid individuals, but not genetic mapping or genome sequencing. To illustrate our approach, we investigated genetic causes for hybrid incompatibility between differentiated lineages of the haplodiploid spider mite Tetranychus evansi, and show that strong, but incomplete, hybrid breakdown occurs. In addition, by comparing the genotypes of viable hybrid males and inviable hybrid male eggs for eight microsatellite loci, we show that nuclear and cytonuclear BDM interactions constitute the basis of hybrid incompatibility in this species. Our approach opens up possibilities to study BDM interactions in non-model taxa, and may give further insight into the genetic mechanisms behind hybrid incompatibility