Use of the fluorescent dye tetramethylrhodamine methyl ester perchlorate for mitochondrial membrane potential assessment in human spermatozoa

Mitochondrial membrane potential (Delta psi m) is an indicator of sperm quality and its evaluation complements the standard semen analysis. The fluorescent dye JC-1 has been widely used to assess sperm Delta psi m; however, some problems have been detected under certain experimental conditions. Another fluorescent compound, tetramethylrhodamine methyl ester perchlorate (TMRM), has been used in somatic cells and bovine spermatozoa but not in human spermatozoa. TMRM accumulates in hyperpolarised mitochondria and the fluorescence intensity of this compound correlates with Delta psi m. Thus, the aim of this study was to evaluate and validate the usefulness of the fluorescent dye TMRM for measuring sperm Delta psi m. The results showed that TMRM accurately detects sperm populations displaying either high or low Delta psi m. Moreover, TMRM was able to measure sperm Delta psi m under the experimental conditions in which JC-1 had previously presented difficulties. Differences in Delta psi m according to sperm and semen quality were properly detected and a positive correlation between Delta psi m and conventional semen parameters was observed. Finally, a positive correlation was found between the Delta psi m measurement by TMRM and by the widely used JC-1. In conclusion, TMRM is a simple, time-effective method, easy to set in laboratories equipped with flow cytometry technology, and can accurately detect changes in Delta psi m with efficiency comparable to JC-1 without its limitations

Long-term survive of Aliarcobacterbutzleri in two models symbiotic interaction with Acanthamoebacastellanii

Aliarcobacter butzleri (formerly known as Arcobacter butzleri) is an emerging food-borne zoonotic pathogen that establishes in vitro endosymbiotic relationships with Acanthamoeba castellanii, a free-living amoeba. Previously, we described that this bacterium acts as an endocytobiont of A. castellanii, surviving for at least 10 days in absence of bacterial replication. Thus, the aim of this study was to evaluate the ability of A. butzleri to survive as a long-term endosymbiont of A. castellanii for 30 days in two models of symbiotic interaction with A. castellanii: (i) endosymbiotic culture followed by gentamicin protection assay and (ii) transwell co-culture assay. The results allow us to conclude that A. butzleri is capable of surviving as an endosymbiont of A. castellanii for at least 30 days, without multiplying, under controlled laboratory conditions. In addition, in the absence of nutrients and as both microorganisms remain in the same culture, separated by semi-permeable membranes, A. castellanii does not promote the survival of A. butzleri, nor does it multiply. Our findings suggest that the greater survival capacity of A. butzleri is associated with their endosymbiont status inside A. castellanii, pointing out the complexity of this type of symbiotic relationship