Oxidative and Nitrosative Stress Detection in Human Sperm Using Fluorescent Probes.

As a natural by-product of mitochondrial respiration, reactive oxygen species (ROS) in sperm play a role in promoting fertilization, by intervening in a series of events. Nevertheless, an abnormal and uncounteracted increase in ROS production leads to oxidative stress (OS) which can, ultimately, culminate in cell death. An established relationship between OS and male infertility highlights the importance of an accurate detection method for ROS content that can be easily implemented and reproduced in any andrology lab. More recently, reactive nitrogen species (RNS) production and subsequent nitrosative stress have also been described. Here we describe the use of fluorescent probes, including some that targeted to the mitochondria due to the coupling of a cation (TPP+), in order to assess the levels of different ROS and RNS in human sperm using flow cytometry and/or fluorescent microscopy. This methodology is user friendly and accurate and can be safely applied in research- and/or clinical-based contexts.

Production and Purification of Phosphatidylinositol Mannosides from Mycobacterium smegmatis Biomass.

Mycobacterium tuberculosis, the etiological agent of tuberculosis, is regarded as the most successful pathogen of humankind and a major threat to global health. The mycobacterial cell wall is vital for cell growth, virulence, and resistance to antibiotics, and thus constitutes a unique target for drug development. To characterize the enzymes catalyzing the synthesis of the cell wall components, considerable amounts of substrates are required. Since many mycobacterial cell wall lipids, particularly phosphatidylinositol mannosides (PIMs), are not commercially available, isolation from cell biomass is the most straightforward way to obtain these compounds. In this study, we optimized a protocol to extract and purify PIM species, in particular Ac1 PIM2 and Ac1 PIM4 , which can be further used for the identification and characterization of target enzymes. PIMs were extracted from Mycobacterium smegmatis mc2 155 ΔPimE using organic solvents, and purified through three consecutive chromatography steps. Thin-layer chromatography (TLC) was used in-between purification steps to evaluate the success of lipid separation, and nuclear magnetic resonance (NMR) was used for product quantification and to assess purity. Typically, from a 60 g batch of M. smegmatis biomass we were able to isolate approximately 9 mg of Ac1 PIM2 and 1.8 mg of Ac1 PIM4 . This is the first time the purification of phosphatidylinositol tetramannoside has been reported. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Growth of M. smegmatis mc2 155 ∆PimE Basic Protocol 2: Extraction of lipids from M. smegmatis mc2 155 ∆PimE Basic Protocol 3: Treatment of the lipid extract for isolation of phospholipids Basic Protocol 4: Isolation of phosphatidylinositol mannosides Basic Protocol 5: Quantification of phosphatidylinositol mannosides.

Mitochondria-Targeted Compounds to Assess and Improve Human Sperm Function.

Significance: Currently 10%-15% of couples in reproductive age face infertility issues. More importantly, male factor contributes to 50% of these cases (either alone or in combination with female causes). Among various reasons, impaired sperm function is the main cause for male infertility. Furthermore, mitochondrial dysfunction and oxidative stress due to increased reactive oxygen species (ROS) production, particularly of mitochondrial origin, are believed to be the main contributors. Recent Advances: Mitochondrial dysfunction, particularly due to increased ROS production, has often been linked to impaired sperm function/quality. For decades, different methods and approaches have been developed to assess mitochondrial features that might correlate with sperm functionality. This connection is now completely accepted, with mitochondrial functionality assessment used more commonly as a readout of sperm functionality. More recently, mitochondria-targeted compounds are on the frontline for both assessment and therapeutic approaches. Critical Issues: In this review, we summarize the current methods for assessing key mitochondrial parameters known to reflect sperm quality as well as therapeutic strategies using mitochondria-targeted antioxidants aiming to improve sperm function in various situations, particularly after sperm cryopreservation. Future Directions: Although more systematic research is needed, mitochondria-targeted compounds definitely represent a promising tool to assess as well as to protect and improve sperm function. Antioxid. Redox Signal. 37, 451-480.

Mitochondrial Functional Assessment in Mammalian Gametes Using Fluorescent Probes.

A positive relationship between mitochondrial functionality and gamete quality, ultimately contributing to fertilization success and normal embryo development has been established for some years now. Both mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) production are major indicators of mitochondrial function, and the need for accurate biomarkers mirroring gamete quality highlights the importance of a precise assessment of mitochondrial quality and function. In this chapter, we discuss the use of some mitochondrial fluorescent probes coupled to flow cytometry and/or fluorescence microscopy to specifically assess mitochondrial ROS production and MMP in both sperm and oocytes. Furthermore, as the distribution/aggregation of mitochondria in the oocyte is of interest to determine its quality, a detailed protocol is also given. These methodologies are easy, accurate and can be safely applied in research- and/or clinical-based contexts.

Fluorescent probes for the detection of reactive oxygen species in human spermatozoa.

Reactive oxygen species (ROS) production is a by-product of mitochondrial activity and is necessary for the acquisition of the capacitated state, a requirement for functional spermatozoa. However, an increase in oxidative stress, due to an abnormal production of ROS, has been shown to be related to loss of sperm function, highlighting the importance of an accurate detection of sperm ROS, given the specific nature of this cell. In this work, we tested a variety of commercially available fluorescent probes to detect ROS and reactive nitrogen species (RNS) in human sperm, to define their specificity. Using both flow cytometry (FC) and fluorescence microscopy (FM), we confirmed that MitoSOX™ Red and dihydroethidium (DHE) detect superoxide anion (as determined using antimycin A as a positive control), while DAF-2A detects reactive nitrogen species (namely, nitric oxide). For the first time, we also report that RedoxSensor™ Red CC-1, CellROX® Orange Reagent, and MitoPY1 seem to be mostly sensitive to hydrogen peroxide, but not superoxide. Furthermore, mean fluorescence intensity (and not percentage of labeled cells) is the main parameter that can be reproducibly monitored using this type of methodology.

Can we induce spermatogenesis in the domestic cat using an in vitro tissue culture approach?

The reduced number of animals in most wild felid populations implies a loss of genetic diversity. The death of juveniles, prior to the production of mature sperm, represents a loss of potential genetic contribution to future populations. Since 2011 mouse testicular organ culture has introduced an alternative mechanism to produce sperm in vitro from immature tissue. However, extension of this technology to other species has remained limited. We have used the domestic cat (Felis catus) as a model for wild felids to investigate spermatogenesis initiation and regulation, with the mouse serving as a control species. Testicular tissue fragments were cultured in control medium or medium supplemented with knockout serum replacement (KSR), AlbuMax, beta-estradiol or AlbuMax plus beta-estradiol. Contrary to expectations, and unlike results obtained in mouse controls, no germ cell differentiation could be detected. The only germ cells observed after six weeks of culture were spermatogonia regardless of the initial stage of tubule development in the donor tissue. Moreover, the number of spermatogonia decreased with time in culture in all media tested, especially in the medium supplemented with KSR, while AlbuMax had a slight protective effect. The combination of AlbuMax and beta-estradiol led to an increase in the area occupied by seminiferous tubules, and thus to an increase in total number of spermatogonial cells. Considering all the media combinations tested the stimulus for felid germ cell differentiation in this type of system seems to be different from the mouse. Studies using other triggers of differentiation and tissue survival factors should be performed to pursue this technology for the genetic diversity preservation in wild felids.

Executive functioning in alcoholics following an mHealth cognitive stimulation program: randomized controlled trial.

BACKGROUND: The consequences of alcohol dependence are severe and may range from physical disease to neuropsychological deficits in several cognitive domains. Alcohol abuse has also been related to brain dysfunction specifically in the prefrontal cortex. Conventional neuropsychological interventions (paper-and-pencil cognitive stimulation training) have a positive effect but are time-consuming, costly, and not motivating for patients. OBJECTIVE: Our goal was to test the cognitive effects of a novel approach to neuropsychological intervention, using mobile technology and serious games, on patients with alcohol dependence. METHODS: The trial design consisted of a two-arm study assessing the cognitive outcomes of neuropsychological intervention with mobile serious games (mHealth) versus control (treatment-as-usual with no neuropsychological intervention) in patients undergoing treatment for alcohol dependence syndrome. Sixty-eight patients were recruited from an alcohol-rehab clinic and randomly assigned to the mHealth (n=33) or control condition (n=35). The intervention on the experimental group consisted of a therapist-assisted cognitive stimulation therapy for 4 weeks on a 2-3 days/week basis. RESULTS: Fourteen patients dropped out of the study. The results of the neuropsychological assessments with the remaining 54 patients showed an overall increase (P<.05) of general cognitive abilities, mental flexibility, psychomotor processing speed, and attentional ability in both experimental (n=26) and control groups (n=28). However, there was a more pronounced improvement (P=.01) specifically in frontal lobe functions from baseline (mean 13.89, SE 0.58) to follow-up (mean 15.50, SE 0.46) in the experimental group but not in the control group. CONCLUSIONS: The overall increase in general cognitive function for both experimental and control groups supports the beneficial role of existing alcohol treatment protocols aimed at minimizing withdrawal symptoms, but the differential improvements observed in frontal lobe functioning supports the use of mobile serious games for neuropsychological stimulation to overcome executive dysfunction in patients with alcohol dependence. This trial was negative on two neuropsychological/cognitive tests, and positive on one. TRIAL REGISTRATION: ClinicalTrials.gov NCT01942954; http://www.clinicaltrials.gov/ct2/show/NCT01942954.