Positive selection in gamete interaction proteins in Carnivora.

The absence of robust interspecific isolation barriers among pantherines, including the iconic South American jaguar (Panthera onca), led us to study molecular evolution of typically rapidly evolving reproductive proteins within this subfamily and related groups. In this study, we delved into the evolutionary forces acting on the zona pellucida (ZP) gamete interaction protein family and the sperm-oocyte fusion protein pair IZUMO1-JUNO across the Carnivora order, distinguishing between Caniformia and Feliformia suborders and anticipating few significant diversifying changes in the Pantherinae subfamily. A chromosome-resolved jaguar genome assembly facilitated coding sequences, enabling the reconstruction of protein evolutionary histories. Examining sequence variability across more than 30 Carnivora species revealed that Feliformia exhibited significantly lower diversity compared to its sister taxa, Caniformia. Molecular evolution analyses of ZP2 and ZP3, subunits directly involved in sperm-recognition, unveiled diversifying positive selection in Feliformia, Caniformia and Pantherinae, although no significant changes were linked to sperm binding. Structural cross-linking ZP subunits, ZP4 and ZP1 exhibited lower levels or complete absence of positive selection. Notably, the fusion protein IZUMO1 displayed prominent positive selection signatures and sites in basal lineages of both Caniformia and Feliformia, extending along the Caniformia subtree but absent in Pantherinae. Conversely, JUNO did not exhibit any positive selection signatures across tested lineages and clades. Eight Caniformia-specific positive selected sites in IZUMO1 were detected within two JUNO-interaction clusters. Our findings provide for the first time insights into the evolutionary trajectories of ZP proteins and the IZUMO1-JUNO gamete interaction pair within the Carnivora order.

Unveiling a novel function of CD9 in surface compartmentalization of oocytes.

Gamete fusion is an indispensable process for bearing offspring. In mammals, sperm IZUMO1-oocyte JUNO recognition essentially carries out the primary step of this process. In oocytes, CD9 is also known to play a crucial role in gamete fusion. In particular, microvilli biogenesis through CD9 involvement appears to be a key event for successful gamete fusion, because CD9-disrupted oocytes produce short and sparse microvillous structures, resulting in almost no fusion ability with spermatozoa. In order to determine how CD9 and JUNO cooperate in gamete fusion, we analyzed the molecular profiles of each molecule in CD9- and JUNO-disrupted oocytes. Consequently, we found that CD9 is crucial for the exclusion of GPI-anchored proteins, such as JUNO and CD55, from the cortical actin cap region, suggesting strict molecular organization of the unique surface of this region. Through distinct surface compartmentalization due to CD9 governing, GPI-anchored proteins are confined to the appropriate fusion site of the oocyte.

Modeling the stability of polygonal patterns of vortices at the poles of Jupiter as revealed by the Juno spacecraft.

From its pole-to-pole orbit, the Juno spacecraft discovered arrays of cyclonic vortices in polygonal patterns around the poles of Jupiter. In the north, there are eight vortices around a central vortex, and in the south there are five. The patterns and the individual vortices that define them have been stable since August 2016. The azimuthal velocity profile vs. radius has been measured, but vertical structure is unknown. Here, we ask, what repulsive mechanism prevents the vortices from merging, given that cyclones drift poleward in atmospheres of rotating planets like Earth? What atmospheric properties distinguish Jupiter from Saturn, which has only one cyclone at each pole? We model the vortices using the shallow water equations, which describe a single layer of fluid that moves horizontally and has a free surface that moves up and down in response to fluid convergence and divergence. We find that the stability of the pattern depends mostly on shielding-an anticyclonic ring around each cyclone, but also on the depth. Too little shielding and small depth lead to merging and loss of the polygonal pattern. Too much shielding causes the cyclonic and anticyclonic parts of the vortices to fly apart. The stable polygons exist in between. Why Jupiter's vortices occupy this middle range is unknown. The budget-how the vortices appear and disappear-is also unknown, since no changes, except for an intruder that visited the south pole briefly, have occurred at either pole since Juno arrived at Jupiter in 2016.

Study on the expression patterns and function of JUNO and CD9 in bovine oocytes during in vitro maturation.

Fertilization proteins JUNO and CD9 play vital roles in sperm-egg fusion, but little is known about their expression patterns during in vitro maturation (IVM) and their function during in vitro fertilization (IVF) of bovine oocytes. In this study, qRT-PCR and immunofluorescence staining were used to detect the mRNA and protein expression levels of JUNO and CD9 genes in bovine oocytes and cumulus cells. Then, fertilization rate of MII oocytes treated with (i) JUNO antibody (1, 5 and 25 µg/ml) or (ii) CD9 antibody (1, 5 and 25 µg/ml) or (iii) CD9 antibody (5 µg/ml) + JUNO antibody (5 µg/ml) were recorded. Our results showed that the mRNA and protein expression levels of JUNO and CD9 genes significantly increased from bovine GV oocytes to MII oocytes, and similar mRNA expression patterns of JUNO and CD9 were also detected in cumulus cells. All groups of oocytes treated with CD9 antibody or JUNO antibody showed significantly decreased fertilization rates (p < .05). Particularly, the fertilization ability of oocytes treated with CD9 antibody (5 µg/ml) + JUNO antibody (5 µg/ml) sharply decreased to 3.48 ± 0.11%. In conclusion, our study revealed the expression levels of JUNO and CD9 genes in oocytes and cumulus cells increased during IVM of bovine oocytes, with JUNO protein playing a major role in the fertilization of bovine oocytes.

Juno Plasma Wave Observations at Ganymede.

The Juno Waves instrument measured plasma waves associated with Ganymede's magnetosphere during its flyby on 7 June, day 158, 2021. Three distinct regions were identified including a wake, and nightside and dayside regions in the magnetosphere distinguished by their electron densities and associated variability. The magnetosphere includes electron cyclotron harmonic emissions including a band at the upper hybrid frequency, as well as whistler-mode chorus and hiss. These waves likely interact with energetic electrons in Ganymede's magnetosphere by pitch angle scattering and/or accelerating the electrons. The wake is accentuated by low-frequency turbulence and electrostatic solitary waves. Radio emissions observed before and after the flyby likely have their source in Ganymede's magnetosphere.

Juno's Close Encounter With Ganymede-An Overview.

The Juno spacecraft has been in orbit around Jupiter since 2016. Two flybys of Ganymede were executed in 2021, opportunities realized by evolution of Juno's polar orbit over the intervening 5 years. The geometry of the close flyby just prior to the 34th perijove pass by Jupiter brought the spacecraft inside Ganymede's unique magnetosphere. Juno's payload, designed to study Jupiter's magnetosphere, had ample dynamic range to study Ganymede's magnetosphere. The Juno radio system was used both for gravity measurements and for study of Ganymede's ionosphere. Remote sensing of Ganymede returned new results on geology, surface composition, and thermal properties of the surface and subsurface.

Ganymede Observations by JunoCam on Juno Perijove 34.

During the Juno Mission's encounter with Ganymede on 7 June 2021, the Juno camera (JunoCam) acquired four images of Ganymede in color. These images covered one-sixth of Ganymede at scales from 840 m to ∼4 km/pixel. Most of this area was only previously imaged by Voyager 1 in 1979, at lower spatial resolution and poorer image quality. No changes were observed over this area of Ganymede in the 42 years since Voyager. JunoCam provided overlapping coverage, from which we developed a digital elevation model of the best-resolved area. A 3 km high dome at the subjovian point was confirmed, 450 km by 750 km. We used the JunoCam images to refine the geologic map of Ganymede in eastern Perrine Regio.

Improvement of Fertilization Capacity and Developmental Ability of Vitrified Bovine Oocytes by JUNO mRNA Microinjection and Cholesterol-Loaded Methyl-ß-Cyclodextrin Treatment.

Vitrification of oocytes is crucial for embryo biotechnologies, germplasm cryopreservation of endangered and excellent female animals, and the fertility of humans. However, vitrification significantly impairs the fertilization ability of oocytes, which significantly limits its widely used application. JUNO protein, a receptor for Izumo1, is involved in sperm-oocyte fusion and is an indispensable protein for mammalian fertilization, and its abundance is susceptible to vitrification. However, it is still unclear how vitrification reduces the fertilization capacity of bovine oocytes by affecting JUNO protein. This study was designed to investigate the effect of vitrification on the abundance and post-translational modifications of JUNO protein in bovine oocytes. Our results showed that vitrification did not alter the amino acid sequence of JUNO protein in bovine oocytes. Furthermore, the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis results showed that vitrification significantly reduced the number and changed the location of disulfide bonds, and increased the number of both phosphorylation and glycosylation sites of JUNO protein in bovine oocytes. Finally, the fertilization capacity and development ability of vitrified oocytes treated with 200 pg JUNO mRNA microinjection and cholesterol-loaded methyl-ß-cyclodextrin (CLC/MßCD) were similar to those of fresh oocytes. In conclusion, our results showed that vitrification of bovine oocytes did not alter the protein sequence of JUNO, but induced post-translational modifications and changed protein abundance. Moreover, the fertilization and development ability of vitrified bovine oocytes were improved by the combination treatment of JUNO mRNA microinjection and CLC/MßCD.

2,4-DCP compromises the fertilization capacity of mouse oocytes.

2,4-DCP (2,4-dichlorophenol) is an environmental estrogen that is ubiquitously distributed in the environment and widely used to produce herbicides and pharmaceutical intermediates. Although 2,4-DCP is suspected to have endocrine disruption, the reproductive toxicity of 2,4-DCP in mammals has not been adequately assessed. In the present study, we examined the effect of 2,4-DCP on the fertility of mouse eggs. The data showed that oral administration of 2,4-DCP (180 mg/kg/day for 7 days) compromises the fertilization rate of mouse oocytes. To further analyze the mechanism by which 2,4-DCP decreases fertilization, the key regulators and events during fertilization of mouse eggs were investigated. We found that the dynamics of cortical granules (CGs) were disrupted by showing the redistribution of CG free domain in 2,4-DCP-administered oocytes. This abnormality perturbed the sperm binding site in the zona pellucida (ZP) and dramatically reduced the number of sperm binding to the ZP of 2,4-DCP-administered oocytes. In addition, the abundance of Juno, a sperm receptor on the egg membrane, was also decreased and its distribution was mislocated in 2,4-DCP-administered oocytes. Finally, we validated that the defects of fertilization participants and events caused by 2,4-DCP might be mediated by the increased level of reactive oxygen species-induced apoptosis of oocytes. Therefore, we demonstrate that 2,4-DCP compromises the fertilization ability of mouse oocytes via inducing oxidative stress.

Computer simulations of Jupiter's deep internal dynamics help interpret what Juno sees.

We describe computer simulations of thermal convection and magnetic field generation in Jupiter's deep interior: that is, its convective dynamo. Results from three different simulations highlight the importance of including the dynamics in the very deep interior, although much of the convection and field generation seems to be confined to the upper part of the interior. A long-debated question is to what depth do Jupiter's zonal winds extend below its surface. Our simulations suggest that, if global latitudinally banded patterns in Jupiter's near-surface magnetic and gravity fields were detected by Juno, NASA's orbiting spacecraft at Jupiter [Bolton S, et al. (2017) Science 356:821-825], they would provide evidence for Jupiter's zonal winds extending deep below the surface. One of our simulations has also maintained, for a couple simulated years, a deep axisymmetric inertial wave, with properties at the surface that depend on the size of the model's small rocky core. If such a wave was detected on Jupiter's surface, its latitudes and oscillation frequency would provide evidence for the existence and size of Jupiter's rocky core.

The combination treatment of cholesterol-loaded methyl-ß-cyclodextrin and methyl-ß-cyclodextrin significantly improves the fertilization capacity of vitrified bovine oocytes by protecting fertilization protein JUNO.

Many experiments show that vitrification significantly reduces the fertilization capacity of mammalian oocytes, restricting the application of vitrified oocytes. It has been proven that the JUNO protein plays a vital role in mammalian oocytes fertilization. However, little information is available about the effects of vitrification on the JUNO protein and the procedure to protect it in bovine oocytes. Here, the present study was designed to investigate the effect of vitrification on the JUNO protein level in bovine oocytes. In this study, MII oocytes were treated with cholesterol-loaded methyl-ß-cyclodextrin (CLC; 0, 10, 15, 20 mM) for 45 min before vitrification and methyl-ß-cyclodextrin (MßCD; 0, 2.25, 4.25, 6.25 mM) for 45 min after thawing (38-39°C). Then, the expression level and function of JUNO protein, cholesterol level in the membrane, the externalization of phosphatidylserine, sperm binding capacity and the developmental ability of vitrified bovine oocytes were examined. Our results showed that vitrification significantly decreased the JUNO protein level, cholesterol level, sperm binding capacity, development ability, and increased the promoter methylation level of the JUNO gene and apoptosis level of bovine oocytes. Furthermore, 15 mM CLC + 4.25 mM MßCD treatment significantly improved the cholesterol level and increased sperm binding and development ability of vitrified bovine oocytes. In conclusion, the combination treatment of cholesterol-loaded methyl-ß-cyclodextrin and methyl-ß-cyclodextrin significantly improves the fertilization capacity of vitrified bovine oocytes by protecting fertilization protein JUNO.

Energetic Oxygen and Sulfur Charge States in the Outer Jovian Magnetosphere: Insights From the Cassini Jupiter Flyby.

On 10 January 2001, Cassini briefly entered into the magnetosphere of Jupiter, en route to Saturn. During this excursion into the Jovian magnetosphere, the Cassini Magnetosphere Imaging Instrument/Charge-Energy-Mass Spectrometer detected oxygen and sulfur ions. While Charge-Energy-Mass Spectrometer can distinguish between oxygen and sulfur charge states directly, only 95.9 ± 2.9 keV/e ions were sampled during this interval, allowing for a long time integration of the tenuous outer magnetospheric (~200 RJ) plasma at one energy. For this brief interval for the 95.9 keV/e ions, 96% of oxygen ions were O+, with the other 4% as O2+, while 25% of the energetic sulfur ions were S+, 42% S2+, and 33% S3+. The S2+/O+ flux ratio was observed to be 0.35 (±0.06 Poisson error).

Probing Jovian Broadband Kilometric Radio Sources Tied to the Ultraviolet Main Auroral Oval With Juno.

Observations of Jovian broadband kilometric (bKOM) radiation and ultraviolet (UV) auroras were acquired with the Waves and Juno-UVS instruments for ∼2 hr over the northern and southern polar regions during Juno's perijoves 4, 5, and 6 passes (PJ4, PJ5, and PJ6). During all six time periods, Juno traversed auroral magnetic field lines connecting to the UV main auroral ovals, matching the estimates of bKOM radio source footprints. The localized bKOM radio sources for the PJ4 north pass map to magnetic field lines having distances of 10 to 12 Jovian radii (R J) at the magnetic equator, whereas the extended bKOM radio sources for the other events map to field lines extending to 20-61 R J. We found the peak bKOM intensities during Juno's potential radio source crossings show positive, negative, and no correlations with the UV main oval brightness and color ratio. Only the positive correlations suggest wave-particle energy transport.

Jovian Injections Observed at High Latitude.

The polar orbit of Juno at Jupiter provides a unique opportunity to observe high-latitude energetic particle injections. We measure energy-dispersed impulsive injections of protons and electrons. Ion injection signatures are just as prevalent as electron signatures, contrary to previous equatorial observations. Included are previously unreported observations of high-energy banded structures believed to be remnants of much earlier injections, where the particles have had time to disperse around Jupiter. A model fit of the injections used to estimate timing fits the shape of the proton signatures better than it does the electron shapes, suggesting that electrons and protons are different in their abilities to escape the injection region. We present ultaviolet observations of Jupiter's aurora and discuss the relationship between auroral injection features and in situ injection events. We find, unexpectedly, that the presence of in situ particle injections does not necessarily result in auroral injection signatures.

High-Energy (>10 MeV) Oxygen and Sulfur Ions Observed at Jupiter From Pulse Width Measurements of the JEDI Sensors.

The Jovian polar regions produce X-rays that are characteristic of very energetic oxygen and sulfur that become highly charged on precipitating into Jupiter's upper atmosphere. Juno has traversed the polar regions above where these energetic ions are expected to be precipitating revealing a complex composition and energy structure. Energetic ions are likely to drive the characteristic X-rays observed at Jupiter (Haggerty et al., 2017, https://doi.org/10.1002/2017GL072866; Houston et al., 2018, https://doi.org/10.1002/2017JA024872; Kharchenko et al., 2006, https://doi.org/10.1029/2006GL026039). Motivated by the science of X-ray generation, we describe here Juno Jupiter Energetic Particle Detector Instrument (JEDI) measurements of ions above 1 MeV and demonstrate the capability of measuring oxygen and sulfur ions with energies up to 100 MeV. We detail the process of retrieving ion fluxes from pulse width data on instruments like JEDI (called "puck's"; Clark, Cohen, et al., 2016, https://doi.org/10.1002/2017GL074366; Clark, Mauk, et al., 2016, https://doi.org/10.1002/2015JA022257; Mauk et al., 2013, https://doi.org/10.1007/s11214-013-0025-3) as well as details on retrieving very energetic particles (>20 MeV) above which the pulse width also saturates.

Comparing Electron Energetics and UV Brightness in Jupiter's Northern Polar Region During Juno Perijove 5.

We compare electron and UV observations mapping to the same location in Jupiter's northern polar region, poleward of the main aurora, during Juno perijove 5. Simultaneous peaks in UV brightness and electron energy flux are identified when observations map to the same location at the same time. The downward energy flux during these simultaneous observations was not sufficient to generate the observed UV brightness; the upward energy flux was. We propose that the primary acceleration region is below Juno's altitude, from which the more intense upward electrons originate. For the complete interval, the UV brightness peaked at ~240 kilorayleigh (kR); the downward and upward energy fluxes peaked at 60 and 700 mW/m2, respectively. Increased downward energy fluxes are associated with increased contributions from tens of keV electrons. These observations provide evidence that bidirectional electron beams with broad energy distributions can produce tens to hundreds of kilorayleigh polar UV emissions.

JUNO/JIRAM's view of Jupiter's H3+ emissions.

The instrument JIRAM (Jovian Infrared Auroral Mapper), on board the NASA spacecraft Juno, is both an imager and a spectrometer. Two distinct detectors are used for imaging and spectroscopy. The imager acquires Jupiter images in two bands, one of which (L band, 3.3-3.6 µm) is devoted to monitor the H3+ emission. The spectrometer covers the spectral region from 2 to 5 µm (average spectral resolution 9 nm) with a 256 pixels slit that can observe the same scene of the L band imager with some delay. JIRAM scientific goals are the exploration of the Jovian aurorae and the planet's atmospheric structure, dynamics and composition. Starting early July 2016 Juno is orbiting around Jupiter. Since then, JIRAM has provided an unprecedented amount of measurements, monitoring both Jupiter's atmosphere and aurorae. In particular, the camera has monitored Jupiter's poles with very high spatial resolution, providing new insights in both its aurorae and the polar dynamic. The main findings obtained by the L imager are detailed pictures of Jupiter's aurorae showing an extremely complex morphology of the H3+ distribution in the main oval and in the moon's footprints. The spectrometer has enabled the measure the distribution of both H3+ concentration and temperature. The analysis of the north auroral region limb observations shows that the peak density of H3+ is above 750 km and that often it is anticorrelated to the temperature, confirming the infrared cooling effect of H3+. This article is part of a discussion meeting issue 'Advances in hydrogen molecular ions: H3+, H5+ and beyond'.

Binding of sperm protein Izumo1 and its egg receptor Juno drives Cd9 accumulation in the intercellular contact area prior to fusion during mammalian fertilization.

Little is known about the molecular mechanisms that induce gamete fusion during mammalian fertilization. After initial contact, adhesion between gametes only leads to fusion in the presence of three membrane proteins that are necessary, but insufficient, for fusion: Izumo1 on sperm, its receptor Juno on egg and Cd9 on egg. What happens during this adhesion phase is a crucial issue. Here, we demonstrate that the intercellular adhesion that Izumo1 creates with Juno is conserved in mouse and human eggs. We show that, along with Izumo1, egg Cd9 concomitantly accumulates in the adhesion area. Without egg Cd9, the recruitment kinetics of Izumo1 are accelerated. Our results suggest that this process is conserved across species, as the adhesion partners, Izumo1 and its receptor, are interchangeable between mouse and human. Our findings suggest that Cd9 is a partner of Juno, and these discoveries allow us to propose a new model of the molecular mechanisms leading to gamete fusion, in which the adhesion-induced membrane organization assembles all key players of the fusion machinery.

Melatonin improves the fertilization ability of post-ovulatory aged mouse oocytes by stabilizing ovastacin and Juno to promote sperm binding and fusion.

STUDY QUESTION: What are the underlying mechanisms of the decline in the fertilization ability of post-ovulatory aged oocytes? SUMMARY ANSWER: Melatonin improves the fertilization ability of post-ovulatory aged oocytes by reducing aging-induced reactive oxygen species (ROS) levels and inhibiting apoptosis and by maintaining the levels and localization of the fertilization proteins, ovastacin and Juno. WHAT IS KNOWN ALREADY: Following ovulation, the quality of mammalian metaphase II oocytes irreversibly deteriorates over time with a concomitant loss of fertilization ability. Melatonin has been found to prevent post-ovulatory oocyte aging and extend the window for optimal fertilization in mice. STUDY DESIGN, SIZE, DURATION: Mouse oocytes were randomly assigned to three groups and aged in vitro for 0, 6, 12 and 24 h, respectively. Increasing concentrations of melatonin (10-9 M, 10-7 M, 10-5 M and 10-3 M) were added to the 24 h aging group. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sperm binding assays, in-vitro fertilization, immunofluorescent staining and western blotting were performed to investigate key regulators and events during fertilization of post-ovulatory aged mouse oocytes. MAIN RESULTS AND THE ROLE OF CHANCE: We found that the actin cap which promotes a cortical granule (CG) free domain is disrupted with a re-distribution of CGs in the subcortex of aged oocytes. Ovastacin, a CG metalloendoprotease, is mis-located and prematurely exocytosed in aged oocytes with subsequent cleavage of the zona pellucida protein ZP2. This disrupts the sperm recognition domain and dramatically reduces the number of sperm binding to the zona pellucida. The abundance of Juno, the sperm receptor on the oocyte membrane, also is reduced in aged oocytes. Exposure of aged oocytes to melatonin significantly elevates in-vitro fertilization rates potentially by rescuing the above age-associated defects of fertilization, and reducing ROS and inhibiting apoptosis. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: We explored the mechanisms of the decline in fertilization ability decline in aged mouse oocytes, in vitro but not in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Our findings may contribute to the development a more efficient method, involving melatonin, for improving IVF success rates. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Natural Science Foundation (31571545) and the Natural Science Foundation of Jiangsu Province (BK20150677). The authors have no conflict of interest to disclose.

Melatonin protects oocyte quality from Bisphenol A-induced deterioration in the mouse.

Bisphenol A (BPA) has been reported to adversely affect the mammalian reproductive system in both sexes. However, the underlying mechanisms regarding how BPA disrupts the mammalian oocyte quality and how to prevent it have not been fully defined. Here, we document that BPA weakens oocyte quality by impairing both oocyte meiotic maturation and fertilization ability. We find that oral administration of BPA (100 µg/kg body weight per day for 7 days) compromises the first polar body extrusion (78.0% vs 57.0%, P<.05) by disrupting normal spindle assembly, chromosome alignment, and kinetochore-microtubule attachment. This defect could be remarkably ameliorated (76.7%, P<.05) by concurrent oral administration of melatonin (30 mg/kg body weight per day for 7 days). In addition, BPA administration significantly decreases the fertilization rate of oocytes (87.2% vs 41.1%, P<.05) by reducing the number of sperm binding to the zona pellucida, which is consistent with the premature cleavage of ZP2 as well as the mis-localization and decreased protein level of ovastacin. Also, the localization and protein level of Juno, the sperm receptor on the egg membrane, are strikingly impaired in BPA-administered oocytes. Finally, we show that melatonin administration substantially elevates the in vitro fertilization rate (63.0%, P<.05) by restoring above defects of fertilization proteins and events, which might be mediated by the improvement of oocyte quality via reduction of ROS levels and inhibition of apoptosis. Collectively, our data reveal that melatonin has a protective action against BPA-induced deterioration of oocyte quality in mice.