In Vivo Monitoring of mRNA Movement in Drosophila Body Wall Muscle Cells Reveals the Presence of Myofiber Domains

Background: In skeletal muscle each muscle cell, commonly called myofiber, is actually a large syncytium containing numerous nuclei. Experiments in fixed myofibers show that mRNAs remain localized around the nuclei in which they are produced. Methodology/Principal Findings: In this study we generated transgenic flies that allowed us to investigate the movement of mRNAs in body wall myofibers of living Drosophila embryos. We determined the dynamic properties of GFP-tagged mRNAs using in vivo confocal imaging and photobleaching techniques and found that the GFP-tagged mRNAs are not free to move throughout myofibers. The restricted movement indicated that body wall myofibers consist of three domains. The exchange of mRNAs between the domains is relatively slow, but the GFP-tagged mRNAs move rapidly within these domains. One domain is located at the centre of the cell and is surrounded by nuclei while the other two domains are located at either end of the fiber. To move between these domains mRNAs have to travel past centrally located nuclei. Conclusions/Significance: These data suggest that the domains made visible in our experiments result from prolonged interactions with as yet undefined structures close to the nuclei that prevent GFP-tagged mRNAs from rapidly moving between the domains. This could be of significant importance for the treatment of myopathies using regenerative cellbase

Validation of the blood gas analyzer for pH measurements in IVF culture medium: Prevent suboptimal culture conditions.

Measurement of pH in IVF-media using the blood gas analyzer (BGA) requires validation, because IVF-media is outside the intended scope of the BGA. To determine whether the Siemens Rapidpoint 500 BGA is suitable for pH measurements in IVF-media this study will validate the BGA and assess its accuracy. In this method comparison study, the pH of over three hundred IVF-media samples was measured with the BGA and a pH electrode (Hanna pH checker). The precision of both the BGA and the pH electrode were excellent (coefficient variation <1.4%). However, the closeness of agreement between measured values of both devices were not equivalent to each other in the tested IVF-media, showing 15% to 85% accordance between devices. The pH measured with the blood gas analyzer was also significantly higher in the tested media, compared to that measured by the pH electrode. One of the tested media did not reach its target pH when it was measured with the BGA, even at 9% CO2. The results show that the validated blood gas analyzer produces excellent results in terms of precision but not in terms of accuracy. Inaccurate measurement may lead to misinterpretation of results and consequently to suboptimal culture conditions. Therefore, each laboratory is encouraged to perform a validation of their BGA

Dynamic in vitro culture of cryopreserved-thawed human ovarian cortical tissue using a microfluidics platform does not improve early folliculogenesis

Current strategies for fertility preservation include the cryopreservation of embryos, mature oocytes or ovarian cortical tissue for autologous transplantation. However, not all patients that could benefit from fertility preservation can use the currently available technology. In this regard, obtaining functional mature oocytes from ovarian cortical tissue in vitro would represent a major breakthrough in fertility preservation as well as in human medically assisted reproduction. In this study, we have used a microfluidics platform to culture cryopreserved-thawed human cortical tissue for a period of 8 days and evaluated the effect of two different flow rates in follicular activation and growth. The results showed that this dynamic system supported follicular development up to the secondary stage within 8 days, albeit with low efficiency. Surprisingly, the stromal cells in the ovarian cortical tissue were highly sensitive to flow and showed high levels of apoptosis when cultured under high flow rate. Moreover, after 8 days in culture, the stromal compartment showed increase levels of collagen deposition, in particular in static culture. Although microfluidics dynamic platforms have great potential to simulate tissue-level physiology, this system still needs optimization to meet the requirements for an efficient in vitro early follicular growth