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Fluorescence probes used for detection of reactive oxygen species

According to the traditional model, steroid hormones bind to intracellular receptors and subsequently modulate transcription and protein synthesis, thus triggering genomic events finally responsible for delayed effects. Based upon similarities in molecular structure, specific receptors for steroids, vitamin D3 derivatives, thyroid hormone, retinoids, and a variety of orphan receptors are considered to represent a superfamily of steroid receptors. In addition, very rapid effects of steroids mainly affecting intracellular signaling have been widely recognized that are clearly incompatible with the genomic model. These rapid, nongenomic steroid actions are likely to be transmitted via specific membrane receptors. Evidence for nongenomic steroid effects and distinct receptors involved is presented for all steroid groups including related compounds like vitamin D3 and thyroid hormones. The physiological and clinical relevance of these rapid effects is still largely unclear, but their existence in vivo has been clearly shown in various settings including human studies. Drugs that specifically affect nongenomic steroid action may find applications in various clinical areas such as cardiovascular and central nervous disorders, electrolyte homeostasis, and infertility. In addition to a short description of genomic steroid action, this review pays particular attention to the current knowledge and important results on the mechanisms of nongenomic steroid action. The modes of action are discussed in relation to their potential physiological or pathophysiological relevance and with regard to a cross-talk between genomic and nongenomic responses.

Multiple Actions of Steroid Hormones—A Focus on Rapid, Nongenomic Effects

At coitus, human sperm are deposited into the anterior vagina, where, to avoid vaginal acid and immune responses, they quickly contact cervical mucus and enter the cervix. Cervical mucus filters out sperm with poor morphology and motility and as such only a minority of ejaculated sperm actually enter the cervix. In the uterus, muscular contractions may enhance passage of sperm through the uterine cavity. A few thousand sperm swim through the uterotubal junctions to reach the Fallopian tubes (uterine tubes, oviducts) where sperm are stored in a reservoir, or at least maintained in a fertile state, by interacting with endosalpingeal (oviductal) epithelium. As the time of ovulation approaches, sperm become capacitated and hyperactivated, which enables them to proceed towards the tubal ampulla. Sperm may be guided to the oocyte by a combination of thermotaxis and chemotaxis. Motility hyperactivation assists sperm in penetrating mucus in the tubes and the cumulus oophorus and zona pellucida of the oocyte, so that they may finally fuse with the oocyte plasma membrane. Knowledge of the biology of sperm transport can inspire improvements in artificial insemination, IVF, the diagnosis of infertility and the development of contraceptives.

/pdf/sperm-transport-in-the-female-reproductive-tract-1wd76o2ooi.pdf

Sperm transport in the female reproductive tract

Cell death can be executed through different subroutines. Since the description of ferroptosis as an iron-dependent form of non-apoptotic cell death in 2012, there has been mounting interest in the process and function of ferroptosis. Ferroptosis can occur through two major pathways, the extrinsic or transporter-dependent pathway and the intrinsic or enzyme-regulated pathway. Ferroptosis is caused by a redox imbalance between the production of oxidants and antioxidants, which is driven by the abnormal expression and activity of multiple redox-active enzymes that produce or detoxify free radicals and lipid oxidation products. Accordingly, ferroptosis is precisely regulated at multiple levels, including epigenetic, transcriptional, posttranscriptional and posttranslational layers. The transcription factor NFE2L2 plays a central role in upregulating anti-ferroptotic defense, whereas selective autophagy may promote ferroptotic death. Here, we review current knowledge on the integrated molecular machinery of ferroptosis and describe how dysregulated ferroptosis is involved in cancer, neurodegeneration, tissue injury, inflammation, and infection.

/pdf/ferroptosis-molecular-mechanisms-and-health-implications-4yk94rluyi.pdf

Ferroptosis: molecular mechanisms and health implications

Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects.

http://xrm.phys.northwestern.edu/research/pdf_papers/2000/flesch_bba2000.pdf

Dynamics of the mammalian sperm plasma membrane in the process of fertilization.

A flow cytometric procedure was used to follow the effect of bicarbonate, a key inducer of sperm capacitation in vitro, on the transbilayer behavior of C6NBD-phospholipids in the plasma membrane of living acrosome-intact boar spermatozoa under physiological conditions. In the absence of bicarbonate, 97% of C6NBD-phosphatidylserine and 78% of C6NBD-phosphatidylethanolamine was rapidly translocated from the outer leaflet to the inner, whereas relatively little C6NBD-phosphatidylcholine and C6NBD-sphingomyelin was translocated (15% and 5%, respectively). Inclusion of 15 mM bicarbonate/5%CO(2) markedly slowed down the rates of translocation of the aminophospholipids without altering their final distribution, whereas it increased the proportions of C6NBD-phosphatidylcholine and C6NBD-sphingomyelin translocated (30% and 20%, respectively). Bicarbonate activated very markedly the outward translocation of all four phospholipid classes. The changes in C6NBD-phospholipid behavior were accompanied by increased membrane lipid disorder as detected by merocyanine 540, and also by increased potential for phospholipase catabolism of the C6NBD-phospholipid probes. All three changes were mediated via a cAMP-dependent protein phosphorylation pathway. We suspect that the changes result from an activation of the non- specific bidirectional translocase ('scramblase'). They have important implications with respect to sperm fertilizing function.

The capacitating agent bicarbonate induces protein kinase A-dependent changes in phospholipid transbilayer behavior in the sperm plasma membrane.

The primary aim of this study was to establish a flow cytometric technique for determining the capacitation status of stallion spermatozoa. To this end, a flow cytometric technique that demonstrates changes in plasma membrane fluidity; namely, merocyanine 540 staining, was compared with the more conventional Ca2+-dependent fluorescence microscopic technique, chlortetracycline (CTC) staining, for assessing capacitation status. In addition, the effect of bicarbonate/CO2 on the progress of capacitation and the acrosome reaction (AR) and on temporal changes in sperm motility, with particular regard to hyperactivation, was analyzed. For the study, fresh semen was washed and then incubated for 5 h in bicarbonate-containing or bicarbonate-free medium, with or without Ca2+ ionophore to induce the AR, and at intervals during incubation aliquots were taken and analyzed for capacitation and acrosome status. The AR was assessed using both the CTC and fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) st...

https://bioone.org/journals/biology-of-reproduction/volume-65/issue-2/biolreprod65.2.462/Evaluation-of-In-Vitro-Capacitation-of-Stallion-Spermatozoa1/10.1095/biolreprod65.2.462.pdf

Evaluation of in vitro capacitation of stallion spermatozoa.

Mammalian sperm cells are activated prior to fertilization by high bicarbonate levels, which facilitate lipoprotein-mediated cholesterol efflux. The role of bicarbonate and cholesterol acceptors on the cholesterol organization in the sperm plasma membrane was tested. Bicarbonate induced an albumin-independent change in lipid architecture that was detectable by an increase in merocyanine staining (due to protein kinase A-mediated phospholipid scrambling). The response was limited to a subpopulation of viable sperm cells that were sorted from the non-responding subpopulation by flow cytometry. The responding cells had reduced cholesterol levels (30% reduction) compared with non-responding cells. The subpopulation differences were caused by variable efficiencies in epididymal maturation as judged by cell morphology. Membrane cholesterol organization was observed with filipin, which labeled the entire sperm surface of non-stimulated and non-responding cells, but labeled only the apical surface area of bicarbonate-responding cells. Addition of albumin caused cholesterol efflux, but only in bicarbonate-responding cells that exhibited virtually no filipin labeling in the sperm head area. Albumin had no effect on other lipid components, and no affinity for cholesterol in the absence of bicarbonate. Therefore, bicarbonate induces first a lateral redistribution in the low cholesterol containing spermatozoa, which in turn facilitates cholesterol extraction by albumin. A model is proposed in which phospholipid scrambling induces the formation of an apical membrane raft in the sperm head surface that enables albumin mediated efflux of cholesterol.

Bicarbonate stimulated phospholipid scrambling induces cholesterol redistribution and enables cholesterol depletion in the sperm plasma membrane

Simultaneously evaluating postthaw viability and acrosome integrity of spermatozoa by flow cytometry would provide a valuable testing tool in both research and routine work. In the present study, a new triple-stain combination was developed for the simultaneous evaluation of viability and acrosome integrity of bovine sperm processed in egg yolk-based extender by flow cytometer. SYBR-14 and propidium iodide (PI) enabled the discrimination of sperm cells from egg yolk and debris particles, which was instrumental for the flow cytometric analyses of frozen-thawed bovine sperm, because it implied that washing steps to remove egg yolk were no longer required. In addition, phycoerythrin-conjugated peanut agglutinin (PE-PNA) was used to discriminate acrosome-damaged/reacted sperm cells from acrosome-intact cells. Repeatability was calculated using two processed ejaculates of 10 bulls. Three straws per batch were analyzed in duplicate measurements. Method-agreement analysis between the SYBR-14/PE-PNA/PI and fluorescein isothiocyanate (FITC)-conjugated PNA was performed, with FITC-PNA/PI staining being carried out on 14 frozen-thawed semen samples immediately after thawing and after a 3-h incubation at 37 degrees C. The British Standards Institution repeatability index of the SYBR-14/PE-PNA/PI combination was 2.6%. On average, the FITC-PNA/PI method showed a 6.3% overestimation of the live and acrosome-intact sperm cell subpopulation. In conclusion, the new triple-stain combination is highly repeatable and easy to use in routine application, and it provides a more precise estimate for the rate of sperm cells with intact head membrane and acrosome compared to the generally used and validated FITC-PNA/PI staining.

A Triple-Stain Flow Cytometric Method to Assess Plasma- and Acrosome-Membrane Integrity of Cryopreserved Bovine Sperm Immediately after Thawing in Presence of Egg-Yolk Particles

Barend M. Gadella

Papers

Dynamics of the mammalian sperm plasma membrane in the process of fertilization.

The capacitating agent bicarbonate induces protein kinase A-dependent changes in phospholipid transbilayer behavior in the sperm plasma membrane.

Evaluation of in vitro capacitation of stallion spermatozoa.

Bicarbonate stimulated phospholipid scrambling induces cholesterol redistribution and enables cholesterol depletion in the sperm plasma membrane

A Triple-Stain Flow Cytometric Method to Assess Plasma- and Acrosome-Membrane Integrity of Cryopreserved Bovine Sperm Immediately after Thawing in Presence of Egg-Yolk Particles