Porcine oocyte preincubation in oviductal fluid flush before <i>in vitro</i> fertilization in the presence of oviductal epithelial cells improves monospermic zygote production

SummaryThe present study was designed to evaluate the effect of the combination of oviduct fluid flush (OFF) and oviduct epithelial cells (OEC) in modulating the incidence of polyspermy in pigs. Therefore, for in vitro fertilization (IVF), oocyte and sperm were co-cultured in Tris-buffered medium (TBM) either supplemented with 10% OFF (OFFD group), or in the presence of a bovine OEC monolayer (OEC group), or the oocytes were exposed to OFF for 30 min before IVF (OFFB group), or in the presence of an OEC monolayer (OFFB + OEC group). Regardless of sperm concentration used (0.5, 1.5, and 4.5 × 105 cells/ml), supplementation of IVF medium with 10% OFF led to an increased (P &lt; 0.05) monospermy rate, without alteration (P &gt; 0.05) of the penetration rate in comparison with the control and OEC groups. When the IVF medium was supplemented with heparin, an overall increase (P &lt; 0.05) of the final output of the IVF system in terms of zygotes with two pronuclei (2PN) was observed in the OFFD group, compared with the control and OEC groups, at a sperm concentration of 4.5 × 105 cells/ml. At this concentration, OFFB improved the monospermy rate but decreased the penetration rate, resulting in low efficiency of monospermic zygotes production. Despite this, no major effect was observed in the developmental competence of the presumed zygotes up to the blastocyst stage. The combination of OFFB with OEC improved the penetration rate, while maintaining the high monospermic rate induced by OFFB. In conclusion, the combination of treatment of oocytes by diluted OFF 30 min before IVF, followed by IVF in the presence of OEC, improved monospermic zygote production without reducing the penetration rate, when the IVF medium was supplemented with heparin.</jats:p

122 Proteome of extracellular vesicles from follicular fluid of bovine 3- to 6-mm follicles: Similarity and specificity compared with granulosa cells

Extracellular vesicles (EVs) are released from cells and are present in most bodily fluids. Small EVs, 30-150nm in diameter, known as exosomes, transport regulatory noncoding RNA, lipids, and proteins, and thus participate in cell-to-cell communications and signalling. Exosomes from bovine ovarian follicular fluid (FF) can stimulate proliferation of granulosa cells (GCs), cellular uptake, and cumulus expansion, and therefore might be useful in reproductive technologies. The aim of our study was to characterise the protein content of bovine FF exosomes and compare it with the GC proteome in order to better understand the origin of these EVs and their possible functions. We extracted EVs from FF of bovine 3- to 6-mm ovarian follicles (enclosed oocytes routinely used for invitro embryo production (IVEP)) via a series of centrifugations at 2,000g to 100 000g. Transmission electron microscopy analysis revealed that 87.9% of extracted EVs had a larger size of exosomes, and 11.8% had smaller size (&amp;lt;30nm); only 0.3% of EVs ranged from 150 to 250nm. For proteome analysis, 80µg of total proteins extracted from EVs and GCs originating from the same follicles were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After trypsin proteolytic digestion, peptide mixtures were analysed via nanoflow liquid chromatography coupled to high-resolution tandem mass spectrometry (MS/MS) using LTQ Orbitrap Velos (Thermo Fisher Scientific). We used Mascot engine (Matrix Science) against the NCBIprot mammalian database to perform MS/MS ion searches; proteins identified with at least two peptides were clustered with Scaffold 4.0 software. We identified 542 protein isoforms that represented 221 and 299 clusters of unique proteins in EVs and GCs, respectively. Of these, 158 are common among EVs and GCs and demonstrate significant enrichment (P&amp;lt;0.01) in Gene Ontology (GO) terms related to protein folding in endoplasmic reticulum, receptor-mediated endocytosis, cell-cell adhesion, translation processes, binding of proteins, RNA or cholesterol, and antioxidant activity. Of common proteins, 69.6% are mapped to the GO “extracellular exosome.” Of the 63 proteins over-represented in EVs, 37 are ribosomal proteins involved in translation, rRNA processing, and mRNA catabolic processes. Of the proteins over-represented in GCs, 141 are mainly involved in carbon, fatty acid, and tricarboxylic acid (TCA) metabolism, oxidation-reduction processes, NAD and poly(A) RNA binding, and cell cycle regulation. These proteins are enriched in GO terms related to membrane, mitochondrion, and extracellular exosomes. Our findings indicate that proliferating GCs of antral follicles actively synthesise and secrete exosomes, which contain significant quantity of different ribosomal proteins. These RNA-binding proteins may serve to compact the cargo of different types of RNAs-both regulatory and designated for degradation, which are transported by FF exosomes. Funds were provided by INRA PHASE France and by the Russian Science Foundation (project 19-16-00115). </jats:p

Heat-shock protein A8 restores sperm membrane integrity by increasing plasma membrane fluidity

The constitutive 70 kDa heat-shock protein, HSPA8, has previously been shown to contribute to the long-term survival of spermatozoa inside the mammalian female reproductive tract. Here, we show that a recombinant form of HSPA8 rapidly promotes the viability of uncapacitated spermatozoa, the ability of spermatozoa to bind to oviductal epithelial cells, enhances IVF performance, and decreases sperm mitochondrial activity. Fluorescence recovery after photobleaching revealed that the repair of membrane damage is achieved by an almost instantaneous increase in sperm membrane fluidity. The ability of HSPA8 to influence membrane stability and fluidity, as well as its conserved nature among mammalian species, supports the idea that this protein protects sperm survival through membrane repair mechanisms.Free Persian abstractA Persian translation of the abstract is freely available online athttp://www.reproduction-online.org/content/147/5/719/suppl/DC1</jats:p

Design of an intake and a thruster for an atmosphere-breathing electric propulsion system

Challenging space missions include those at very low altitudes, where the atmosphere is the source of aerodynamic drag on the spacecraft, that finally defines the mission's lifetime, unless a way to compensate for it is provided. This environment is named Very Low Earth Orbit (VLEO) and it is defined for h < 450km. In addition to the spacecraft's aerodynamic design, to extend the lifetime of such missions, an efficient propulsion system is required. One solution is Atmosphere-Breathing Electric Propulsion (ABEP), in which the propulsion system collects the atmospheric particles to be used as propellant for an electric thruster. The system could remove the requirement of carrying propellant on-board, and could also be applied to any planetary body with atmosphere, enabling new missions at low altitude ranges for longer missions' duration. One of the objectives of the H2020 DISCOVERER project, is the development of an intake and an electrode-less plasma thruster for an ABEP system. This article describes the characteristics of intake design and the respective final designs based on simulations, providing collection efficiencies up to 94%. Furthermore, the radio frequency (RF) Helicon-based plasma thruster (IPT) is hereby presented as well, while its performances are being evaluated, the IPT has been operated with single atmospheric species as propellant, and has highlighted very low input power requirement for operation at comparable mass flow rates P similar to 60w.SP