Seminal extracellular vesicles (EVs) include exosomes (ø 40-120 nm) and microvesicles (MVs, ø 120-1000 nm), which would be involved in multiple functional reproductive roles. The study aimed to ...establish which EV subtypes are present in pig semen, using a high-resolution flow cytometer to explore differences in their tetraspanin expression profile. The EVs were isolated from 12 pig ejaculates using serial ultracentrifugation and characterized by dynamic light scattering and electron microscopy for size and morphology as well as for tetraspanin expression using flow cytometry with Carboxyfluorescein succinimidyl ester (CFSE) and antibodies against CD9, CD63 and CD81. Pig semen contained a heterogeneous EV-population regarding size and morphology. Flow cytometric analysis demonstrated that the proportion of EVs expressing CD63 and CD9 was higher in MVs (P < 0.001 and P < 0.05, respectively) than in exosomes, while the opposite was true for CD81; higher (P < 0.001) in exosomes than in MVs. In conclusion, (1) the new generation of flow cytometers are able to accurately identify EVs and to gate them in two size-different populations named exosomes and MVs. (2) Tetraspanins CD9, CD63 and CD81 are present in both seminal EVs, albeit with exosomes and MVs differing in expression profiles, suggesting dissimilar cargo and binding affinity.
Artificial insemination with cryopreserved spermatozoa is a major assisted reproductive technology in many species. In horses, as in humans, insemination with cryopreserved sperm is associated with ...lower pregnancy rates than those for fresh sperm, however, direct effects of sperm cryopreservation on the development of resulting embryos are largely unexplored. The aim of this study was to investigate differences in gene expression between embryos resulting from fertilization with fresh or cryopreserved sperm. Embryos were obtained at 8, 10 or 12 days after ovulation from mares inseminated post-ovulation on successive cycles with either fresh sperm or frozen-thawed sperm from the same stallion, providing matched embryo pairs at each day. RNA was isolated from two matched pairs (4 embryos) for each day, and cDNA libraries were built and sequenced. Significant differences in transcripts per kilobase million (TPM) were determined using (i) genes for which the expression difference between treatments was higher than 99% of that in the random case (P < 0.01), and (ii) genes for which the fold change was ≥ 2, to avoid expression bias in selection of the candidate genes. Molecular pathways were explored using the DAVID webserver, followed by network analyses using STRING, with a threshold of 0.700 for positive interactions. The transcriptional profile of embryos obtained with frozen-thawed sperm differed significantly from that for embryos derived from fresh sperm on all days, showing significant down-regulation of genes involved in biological pathways related to oxidative phosphorylation, DNA binding, DNA replication, and immune response. Many genes with reduced expression were orthologs of genes known to be embryonic lethal in mice. This study, for the first time, provides evidence of altered transcription in embryos resulting from fertilization with cryopreserved spermatozoa in any species. As sperm cryopreservation is commonly used in many species, including human, the effect of this intervention on expression of developmentally important genes in resulting embryos warrants attention.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Seminal Plasma: Relevant for Fertility? Rodriguez-Martinez, Heriberto; Martinez, Emilio A; Calvete, Juan J ...
International journal of molecular sciences,
04/2021, Letnik:
22, Številka:
9
Journal Article
Recenzirano
Odprti dostop
Seminal plasma (SP), the non-cellular component of semen, is a heterogeneous composite fluid built by secretions of the testis, the epididymis and the accessory sexual glands. Its composition, ...despite species-specific anatomical peculiarities, consistently contains inorganic ions, specific hormones, proteins and peptides, including cytokines and enzymes, cholesterol, DNA and RNA-the latter often protected within epididymis- or prostate-derived extracellular vesicles. It is beyond question that the SP participates in diverse aspects of sperm function pre-fertilization events. The SP also interacts with the various compartments of the tubular genital tract, triggering changes in gene function that prepares for an eventual successful pregnancy; thus, it ultimately modulates fertility. Despite these concepts, it is imperative to remember that SP-free spermatozoa (epididymal or washed ejaculated) are still fertile, so this review shall focus on the differences between the in vivo roles of the SP following semen deposition in the female and those regarding additions of SP on spermatozoa handled for artificial reproduction, including cryopreservation, from artificial insemination to in vitro fertilization. This review attempts, including our own results on model animal species, to critically summarize the current knowledge of the reproductive roles played by SP components, particularly in our own species, which is increasingly affected by infertility. The ultimate goal is to reconcile the delicate balance between the SP molecular concentration and their concerted effects after temporal exposure in vivo. We aim to appraise the functions of the SP components, their relevance as diagnostic biomarkers and their value as eventual additives to refine reproductive strategies, including biotechnologies, in livestock models and humans.
Rural areas particularly suffer from economic crises where they overlap with long‐term negative effects of climate change. Here, we introduce the concept of mycotourism by means of mushroom‐related ...industries in central‐northeastern Spain. We describe how this novel branch of eco‐tourism can help stabilize social and political structures. Likewise, we illuminate the potential of mycotourism to compensate for some losses related to widespread unemployment and summer drought, as well as to generate unexpectedly fruitful research opportunities. Focussing on Spain's emerging black truffle industry, we recommend a stronger involvement of natural sciences, conservation services, and management strategies in commercial endeavors. We emphasize the relevance of direct and indirect climatic impacts on ecological and societal systems as well as on economic markets. Moreover, we stress the importance of a vital science–policy interface at various scales, with immediate opportunities for sustainable landscape protection and the preservation of biological diversity.
Global warming is pushing populations outside their range of physiological tolerance. According to the environmental envelope framework, the most vulnerable populations occur near the climatic edge ...of their species' distributions. In contrast, populations from the climatic center of the species range should be relatively buffered against climate warming. We tested this latter prediction using a combination of linear mixed effects and machine learning algorithms on an extensive, citizen‐scientist generated dataset on the fruitbody productivity of the Burgundy (aka summer) truffle (Tuber aestivum Vittad.), a keystone, ectomycorrhizal tree‐symbiont occurring on a wide range of temperate climates. T. aestivum's fruitbody productivity was monitored at 3‐week resolution over up to 8 continuous years at 20 sites distributed in the climatic center of its European distribution in southwest Germany and Switzerland. We found that T. aestivum fruitbody production is more sensitive to summer drought than would be expected from the breadth of its species' climatic niche. The monitored populations occurring nearly 5°C colder than the edge of their species' climatic distribution. However, interannual fruitbody productivity (truffle mass year−1) fell by a median loss of 22% for every 1°C increase in summer temperature over a site's 30‐year mean. Among the most productive monitored populations, the temperature sensitivity was even higher, with single summer temperature anomalies of 3°C sufficient to stop fruitbody production altogether. Interannual truffle productivity was also related to the phenology of host trees, with ~22 g less truffle mass for each 1‐day reduction in the length of the tree growing season. Increasing summer drought extremes are therefore likely to reduce fruiting among summer truffle populations throughout Central Europe. Our results suggest that European T. aestivum may be a mosaic of vulnerable populations, sensitive to climate‐driven declines at lower thresholds than implied by its species distribution model.
We worked with citizen scientists to monitor the productivity of the widely distributed summer truffle (Tuber aestivum) over eight years. We found that hot, dry summers substantially reduce truffle yields. Populations from the center of the species' range cannot tolerate conditions experienced by populations at the hotter extremes, suggesting local adaptation. This may exacerbate summer truffles response to climate change, sensitizing populations to the increasing the frequency of summer drought.
Some stallions yield ejaculates that do not tolerate conservation by refrigeration prior to artificial insemination (AI), showing improvement after removal of most of the seminal plasma (SP) by ...centrifugation. In this study, the SP-proteome of 10 different stallions was defined through high-performance liquid chromatography with tandem mass spectrometry and bioinformatic analysis in relation to the ability of the ejaculates to maintain semen quality when cooled and stored at 5°C. Stallions were classified into three groups, depending on this ability: those maintaining good quality after direct extension in a commercial extender (good), stallions requiring removal of seminal plasma (RSP) to maintain seminal quality (good-RSP), and stallions, unable to maintain good semen quality even after RSP (poor). Pathway enrichment analysis of the proteins identified in whole equine SP using human orthologs was performed using g: profiler showing enriched Reactome and the Kyoto Encyclopedia of Genes and Genomes pathways related to hexose metabolism, vesicle mediated transport, post translational modification of proteins and immune response. Specific proteins overrepresented in stallions tolerating conservation by refrigeration included a peroxiredoxin-6 like protein, and transcobalamin-2, a primary vitamin B12-binding, and transport protein. Also, the protein involved in protein glycosylation, ST3 beta-galactoside alpha-2,3-sialyltransferase 1 was present in good stallions. These proteins were nearly absent in poor stallions. Particularly, annexinA2 appeared as to be the most powerful discriminant variable for identification of stallions needing RSP prior to refrigeration, with a P = 0.002 and a q value = 0.005. Overall this is the first detailed study of the equine SP-proteome, showing the potential value of specific proteins as discriminant bio-markers for clinical classification of stallions for AI. Summary sentence The seminal plasma protein Annexin A2 identifies ejaculates needing removal of seminal plasma prior to conservation in refrigeration.
The unprecedented price inflation of Black truffles, recently exceeding 5000 Euro kg−1 (in Zurich), is a combined result of increasing global demands and decreasing Mediterranean harvests. Since the ...effects of long-term irrigation and climate variation on symbiotic fungus-host interaction and the development of belowground microbes are poorly understood, the establishment and maintenance of truffle plantations remains a risky venture. Using 49 years of continuous harvest and climate data from Spain, France and Italy, we demonstrate how truffle production rates, between November and March, significantly rely on previous June-August precipitation totals, whereas too much autumnal rainfall affects the subsequent winter harvest negatively. Despite a complex climate-host-fungus relationship, our findings show that southern European truffle yields can be predicted at highest probability (r = 0.78, t-stat = 5.645, prob = 0.000 01). Moreover, we demonstrate the reliability of national truffle inventories since 1970, and question the timing and dose of many of the currently operating irrigation systems. Finally, our results suggest that Black truffle mycorrhizal colonization of host fine roots, the sexualisation of mycelium, and the formation of peridium are strongly controlled by natural summer rainfall. Recognising the drought-vulnerability of southern Europe's rapidly growing truffle sector, we encourage a stronger liaison between farmers, politicians and scientists to maintain ecological and economic sustainability under predicted climate change in the Mediterranean basin.
Abstract
Climate change has been described as the main threat for the cultivation and growth of truffles, but hydroclimate variability and model uncertainty challenge regional projections and ...adaptation strategies of the emerging sector. Here, we conduct a literature review to define the main Périgord truffle growing regions around the world and use 20 global climate models to assess the impact of future trends and extremes in temperature, precipitation and soil moisture on truffle production rates and price levels in all cultivation regions in the Americas, Europe, South Africa, and Australasia. Climate model simulations project 2.3 million km
2
of suitable land for truffle growth will experience 50% faster aridification than the rests of the global land surface, with significantly more heat waves between 2070 and 2099 CE. Overall, truffle production rates will decrease by ∼15%, while associated price levels will increase by ∼36%. At the same time, a predicted increase in summer precipitation and less intense warming over Australasia will likely alleviate water scarcity and support higher yields of more affordable truffles. Our findings are relevant for truffle farmers and businesses to adapt their irrigation systems and management strategies to future climate change.
Oxidative stress has been linked to sperm death and the accelerated senescence of cryopreserved spermatozoa. However, the molecular mechanisms behind this phenomenon remain poorly understood. ...Reactive oxygen species (ROS) are considered relevant signaling molecules for sperm function, only becoming detrimental when ROS homeostasis is lost. We hereby hypothesize that a major component of the alteration of ROS homeostasis in cryopreserved spermatozoa is the exhaustion of intrinsic antioxidant defense mechanisms. To test this hypothesis, semen from seven stallions was frozen using a standard technique. The parameters of sperm quality (motility, velocity, and membrane integrity) and markers of sperm senescence (caspase 3, 4-hydroxynonenal, and mitochondrial membrane potential) were assessed before and after cryopreservation. Changes in the intracellular thiol content were also monitored. Cryopreservation caused significant increases in senescence markers as well as dramatic depletion of intracellular thiols to less than half of the initial values (P < 0.001) postthaw. Interestingly, very high and positive correlations were observed among thiol levels with sperm functionality postthaw: total motility (r = 0.931, P < 0.001), progressive motility (r = 0.904, P < 0.001), and percentage of live spermatozoa without active caspase 3 (r = 0.996, P < 0.001). In contrast, negative correlations were detected between active caspase 3 and thiol content both in living (r = -0.896) and dead (r = -0.940) spermatozoa; additionally, 4-hydroxynonenal levels were negatively correlated with thiol levels (r = -0.856). In conclusion, sperm functionality postthaw correlates with the maintenance of adequate levels of intracellular thiols. The accelerated senescence of thawed spermatozoa is related to oxidative and electrophilic stress induced by increased production of 4-hydroxynoneal in thawed samples once intracellular thiols are depleted.
Oxidative stress is considered a major mechanism causing sperm damage during cryopreservation and storage, and underlies male factor infertility. Currently, oxidative stress is no longer believed to ...be caused only by the overproduction of reactive oxygen species, but rather by the deregulation of redox signaling and control mechanisms. With this concept in mind, here, we describe for the first time the presence of the soluble carrier family 7 member 11 (SLC7A11) antiporter, which exchanges extracellular cystine (Cyss) for intracellular glutamate, in stallion spermatozoa, as well as its impact on sperm function using the specific inhibitor sulfasalazine. Spermatozoa incubated with Cyss exhibited an increased intracellular GSH content compared with controls (P < 0.01): 50% in fresh extended stallion spermatozoa and 30% in frozen-thawed spermatozoa. This effect was prevented by the addition of sulfasalazine to the media. Cystine supplementation also reduced the oxidation–reduction potential of spermatozoa, with sulfasalazine only preventing this effect on fresh spermatozoa that were incubated for 3 h at 37°C, but not in frozen-thawed spermatozoa. While sulfasalazine reduced the motility of frozen-thawed spermatozoa, it increased motility in fresh samples. The present findings provide new and relevant data on the mechanism regulating the redox status of spermatozoa and suggest that a different redox regulatory mechanism exists in cryopreserved spermatozoa, thus providing new clues to improve current cryopreservation technologies and treat male factor infertility. Summary Sentence The SLC7A11 antiporter that exchanges cystine by intracellular glutamate is present and functional in stallion spermatozoa, but cryopreserved spermatozoa may present altered functionality.
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