miRNAs as potential spermatogenesis biomarkers in patients with non-obstructive azoospermia

Non-obstructive azoospermia (NOA) is the most severe diagnosis in cases of male infertility. In some cases of NOA a sperm retrieval can be done by microdissection (microTESE) in order to fertilize oocytes through intracytoplasmic sperm injection (ICSI). However, there is a lack of possible biomarkers for non-invasive diagnosis before the microTESE. The microRNAs (miRNAs) are non-coding RNAs with an essential role in post-transcriptional regulation. They play a great role as regulators of various biological processes, including spermatogenesis. Therefore, the number of studies that seek miRNAs as biomarkers (either of infertility or of spermatogenesis in patients with NOA) is increasing markedly

The small molecule raptinal can simultaneously induce apoptosis and inhibit PANX1 activity

Discovery of new small molecules that can activate distinct programmed cell death pathway is of significant interest as a research tool and for the development of novel therapeutics for pathological conditions such as cancer and infectious diseases. The small molecule raptinal was discovered as a pro-apoptotic compound that can rapidly trigger apoptosis by promoting the release of cytochrome c from the mitochondria and subsequently activating the intrinsic apoptotic pathway. As raptinal is very effective at inducing apoptosis in a variety of different cell types in vitro and in vivo, it has been used in many studies investigating cell death as well as the clearance of dying cells. While examining raptinal as an apoptosis inducer, we unexpectedly identified that in addition to its pro-apoptotic activities, raptinal can also inhibit the activity of caspase-activated Pannexin 1 (PANX1), a ubiquitously expressed transmembrane channel that regulates many cell death-associated processes. By implementing numerous biochemical, cell biological and electrophysiological approaches, we discovered that raptinal can simultaneously induce apoptosis and inhibit PANX1 activity. Surprisingly, raptinal was found to inhibit cleavage-activated PANX1 via a mechanism distinct to other well-described PANX1 inhibitors such as carbenoxolone and trovafloxacin. Furthermore, raptinal also interfered with PANX1-regulated apoptotic processes including the release of the 'find-me' signal ATP, the formation of apoptotic cell-derived extracellular vesicles, as well as NLRP3 inflammasome activation. Taken together, these data identify raptinal as the first compound that can simultaneously induce apoptosis and inhibit PANX1 channels. This has broad implications for the use of raptinal in cell death studies as well as in the development new PANX1 inhibitors

Impact of equilibration duration combined with temperature on the outcome of bovine oocyte vitrification

Acord transformatiu CRUE-CSICThe cryopreservation of mammalian oocytes and embryos has become an integral part of assisted reproduction in both humans and veterinary species. However, the methods used to cryopreserve bovine oocytes still have significant shortcomings. A wide variety of approaches has been used to try to improve and optimize methods of cryopreservation. However, these procedures employed are not always designed to specifically take account of the osmotic tolerance response of the cells according to the temperature and time of cryoprotectant (CPA) addition. When these properties are considered, optimal procedures for the addition of CPAs can be designed proactively. Based on in silico and in vitro osmotic observations, we propose shorter dehydration-based protocols at different temperatures (25°C vs. 38.5°C) towards defining an improved cryopreservation method. In vitro matured oocytes were exposed to equilibration solution (ES) at 25°C and 38.5°C and effects of optimized exposure times for each temperature were determined prior to vitrification/warming on oocyte spindle configuration, DNA fragmentation, and further embryo development. Upon exposure to standard ES (7.5% dimethyl sulfoxide + 7.5% ethylene glycol in TCM199 medium + 20% fetal bovine serum), original oocyte volume was recovered within 2 min 30 s at 38.5°C and 5 min 30 s at 25°C. In vitro matured oocytes were then exposed to the aforementioned cryoprotectants at both temperature/duration conditions and vitrified/warmed. While similar percentages of oocytes exhibiting a normally configured spindle and DNA fragmentation were observed in the fresh control group and oocytes vitrified at 38.5°C, significantly higher apoptosis rate and lower percentages of normal spindle configuration were observed in oocytes vitrified at 25°C when compared to control fresh oocytes. Similar cleavage rates and blastocyst yields were observed in the vitrified/38.5°C and fresh controls, while these rates were lower in vitrified/25°C. These results revealed that the limitation of the exposure time of the oocytes to the ES to the point of osmotic equilibrium volume recovery could be a more efficient approach to prepare them for vitrification. Therefore, exposure time to ES to 2 min 30 s at 38.5 °C appears to improve the quality of vitrified/warmed oocytes by protecting spindle integrity and reducing DNA fragmentation thus improving blastocyst rates and embryo quality

The Role of Aquaporin 7 in the Movement of Water and Cryoprotectants in Bovine In Vitro Matured Oocytes

Altres ajuts: Generalitat de Catalunya 2017 SGR 1229The permeability of the plasma membrane to water and cryoprotectants is a critical factor in the effective vitrification of oocytes. The goal of this study is to better understand the pathways used to transport water and other cryoprotectants through the plasma membrane of bovine in vitro matured oocytes, with a focus on the role of aquaporin 7 (AQP7). We demonstrated that cryoprotectants stimulated AQP3 and AQP7 but not AQP9 expression in mature bovine oocytes. Dimethyl sulfoxide upregulates AQP3 expression, while ethylene glycol upregulates AQP7 expression in oocytes in a CPA-dependent fashion. We also demonstrated that exogenous expression of aquaglyceroporins such as AQP7 is possible in in vitro matured oocytes. When permeability values for membrane transport of dimethyl sulfoxide, ethylene glycol and sucrose were assessed, we observed that AQP7 overexpressed oocytes are more permeable to water in the presence of dimethyl sulfoxide solution. These biophysical characteristics, together with the use of membrane transport modeling, will allow re-evaluation and possibly improvement of previously described protocols for bovine oocyte cryopreservation. Aquaglyceroporins are known as channel proteins, and are able to transport water and small neutral solutes. In this study, we evaluate the effect of exposure of in vitro matured bovine oocytes to hyperosmotic solutions containing ethylene glycol (EG), dimethyl sulfoxide (MeSO) or sucrose on the expression levels of AQP3, AQP7 and AQP9. Moreover, we studied whether artificial protein expression of AQP7 in bovine oocytes increases their permeability to water and cryoprotectants. Exposure to hyperosmotic solutions stimulated AQP3 and AQP7 but not AQP9 expression. Oocytes exposed to hyperosmotic MeSO solution exhibited upregulated AQP3 expression, while AQP7 expression was upregulated by EG hyperosmotic exposure. Microinjection of oocytes at the germinal vesicle stage with enhanced green fluorescent protein (EGFP) or EGFP+AQP7 cRNAs resulted in the expression of the corresponding proteins in ≈86% of the metaphase-II stage oocytes. AQP7 facilitated water diffusion when bovine MII oocytes were in presence of MeSO solution but not EG or sucrose solution. However, the overexpression of this aquaporin did not increase membrane permeability to MeSO or EG. In summary, cryoprotectant-induced increase of AQP3 and AQP7 expression could be one of the mechanisms underlying oocyte tolerance to hyperosmotic stress. Water diffusion appears to be improved when AQP7 overexpressed oocytes are exposed to MeSO, shortening the time required for oocytes to achieve osmotic balance with cryoprotectant solutions

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

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