Successful pregnancy requires an appropriate communication between the mother and the embryo. Recently, exosomes and microvesicles, both membrane-bound extracellular vesicles (EVs) present in the oviduct fluid have been proposed as key modulators of this unique cross-talk. However, little is known about their content and their role during oviduct-embryo dialog. Given the known differences in secretions by and oviduct epithelial cells (OEC), we aimed at deciphering the oviduct EVs protein content from both sources. Moreover, we analyzed their functional effect on embryo development. Our study demonstrated for the first time the substantial differences between and oviduct EVs secretion/content. Mass spectrometry analysis identified 319 proteins in EVs, from which 186 were differentially expressed when and EVs were compared ( < 0.01). Interestingly, 97 were exclusively expressed in EVs, 47 were present only in and 175 were common. Functional analysis revealed key proteins involved in sperm-oocyte binding, fertilization and embryo development, some of them lacking in EVs. Moreover, we showed that-produced embryos were able to internalize EVs during culture with a functional effect in the embryo development. EVs increased blastocyst rate, extended embryo survival over time and improved embryo quality. Our study provides the first characterization of oviduct EVs, increasing our understanding of the role of oviduct EVs as modulators of gamete/embryo-oviduct interactions. Moreover, our results point them as promising tools to improve embryo development and survival under conditions.

After insemination in the cow, a sperm reservoir is formed within the oviducts, allowing the storage and then progressive release of spermatozoa toward the ovulated oocyte. In order to investigate the hormonal regulation of these events , the ovarian steroids 17β-estradiol (E2) and progesterone (P4) were added at various concentrations to monolayers of bovine oviduct epithelial cells (BOEC) before or during co-incubation with spermatozoa. Main findings demonstrate that (1) a 18-h pretreatment of BOEC with 100 pg/mL and 100 ng/mL of E2 decreased by 25% the ability of BOEC to bind spermatozoa after 10 min, and for the highest dose of E2, 60 min of co-incubation; (2) P4 at concentrations of 10, 100 and 1000 ng/mL induced the release within 60 min of 32-47% of bound spermatozoa from BOEC; this sperm-releasing effect was maintained after a 18-h pretreatment of BOEC with 100 pg/mL of E2; (3) E2 in concentrations above 100 pg/mL inhibited the releasing effect of P4 on bound sperm in a dose-dependent manner; (4) spermatozoa bound to BOEC, then released from BOEC by the action of P4-induced higher cleavage and blastocyst rates after fertilization than the control group. These results support the hypothesis that the dynamic changes in steroid hormones around the time of ovulation regulate the formation of the sperm reservoir and the timed delivery of capacitated spermatozoa to the site of fertilization.

In vivo monitoring of reactive oxygen species (ROS) in tumors during treatment with anticancer therapy is important for understanding the mechanism of action and in the design of new anticancer drugs. In this work, a platinized nanoelectrode is placed into a single cell for detection of the ROS signal, and drug-induced ROS production is then recorded. The main advantages of this method are the short incubation time with the drug and its high sensitivity which allows the detection of low intracellular ROS concentrations. We have shown that our new method can measure the ROS response to chemotherapy in tumor-bearing mice in realtime. ROS levels were measured in vivo inside the tumor at different depths in response to doxorubicin. This work provides an effective new approach for the measurement of intracellular ROS by platinized nanoelectrodes.

Iron oxide nanoparticles have attracted a great deal of research interest and have been widely used in bioscience and clinical research including as contrast agents for magnetic resonance imaging, hyperthermia and magnetic field assisted radionuclide therapy. It is therefore important to develop methods, which can provide high-throughput screening of biological responses that can predict toxicity. The use of nanoelectrodes for single cell analysis can play a vital role in this process by providing relatively fast, comprehensive, and cost-effective assessment of cellular responses. We have developed a new method for in vitro study of the toxicity of magnetic nanoparticles (NP) based on the measurement of intracellular reactive oxygen species (ROS) by a novel nanoelectrode. Previous studies have suggested that ROS generation is frequently observed with NP toxicity. We have developed a stable probe for measuring intracellular ROS using platinized carbon nanoelectrodes with a cavity on the tip integrated into a micromanipulator on an upright microscope. Our results show a significant difference for intracellular levels of ROS measured in HEK293 and LNCaP cancer cells before and after exposure to 10 nm size iron oxide NP. These results are markedly different from ROS measured after cell incubation with the same concentration of NP using standard methods where no differences have been detected. In summary we have developed a label-free method for assessing nanoparticle toxicity using the rapid (less than 30 minutes) measurement of ROS with a novel nanoelectrode.

High polyspermy is one of the major limitations of porcine invitro fertilisation (IVF). The addition of oviductal fluid (OF) during IVF reduces polyspermy without decreasing the fertilisation rate. Because extracellular vesicles (EVs) have been described as important OF components, the aim of this study was to evaluate the effect of porcine oviductal EVs (poEVs) on IVF efficiency compared with porcine OF (fresh and lyophilised). OF was collected from abattoir oviducts by phosphate-buffered saline flush, and poEVs were isolated by serial ultracentrifugation. Four IVF treatments were conducted: poEVs (0.2mgmL–1), OF (10%), lyophilized and reconstituted pure OF (LOF; 1%) and IVF without supplementation (control). Penetration, monospermy and IVF efficiency were evaluated. Transmission electron microscopy showed an EVs population primarily composed of exosomes (83%; 30–150nm). Supplementation with poEVs during IVF increased monospermy compared with control (44% vs 17%) while maintaining an acceptable penetration rate (61% vs 78% respectively) in a similar way to OF and LOF. Western blotting revealed poEVs proteins involved in early reproductive events, including zona pellucida hardening. In conclusion, our finding show that poEVs are key components of porcine OF and may play roles in porcine fertilisation and polyspermy regulation, suggesting that supplementation with poEVs is a reliable strategy to decrease porcine polyspermy and improve invitro embryo production outcomes.

High-quality, 25 nm octahedral-shaped Fe3O4 magnetite nanocrystals are epitaxially grown on 9 nm Au seed nanoparticles using a modified wet-chemical synthesis. These Fe3O4-Au Janus nanoparticles exhibit bulk-like magnetic properties. Due to their high magnetization and octahedral shape, the hybrids show superior in vitro and in vivo T2 relaxivity for magnetic resonance imaging as compared to other types of Fe3O4-Au hybrids and commercial contrast agents. The nanoparticles provide two functional surfaces for theranostic applications. For the first time, Fe3O4-Au hybrids are conjugated with two fluorescent dyes or the combination of drug and dye allowing the simultaneous tracking of the nanoparticle vehicle and the drug cargo in vitro and in vivo. The delivery to tumors and payload release are demonstrated in real time by intravital microscopy. Replacing the dyes by cell-specific molecules and drugs makes the Fe3O4-Au hybrids a unique all-in-one platform for theranostics.

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We report herein the design, synthesis, and biological investigation of a series of novel Pt­(IV) prodrugs with non-steroidal anti-inflammatory drugs naproxen, diclofenac, and flurbiprofen, as well as these with stearic acid in the axial position. Six Pt­(IV) prodrugs 5–10 were designed, which showed superior antiproliferative activity compared to cisplatin as well as an ability to overcome tumor cell line resistance to cisplatin. By tuning the drug lipophilicity via variation of the axial ligands, the most potent Pt­(IV) prodrug 7 was obtained, with an enhanced cellular accumulation of up to 153-fold that of cisplatin and nanomolar cytotoxicity both in 2D and 3D cell cultures. Pt2+ species were detected at different depths of MCF-7 spheroids after incubation with Pt­(IV) prodrugs using a Pt-coated carbon nanoelectrode. Cisplatin accumulation in vivo in the murine mammary EMT6 tumor tissue of BALB/c mice after Pt­(IV) prodrug injection was proved electrochemically as well. The drug tolerance study on BALB/c mice showed good tolerance of 7 in doses up to 8 mg/kg.