Periodic Reporting for period 1 - SPARTEVs (Impact of extracellular vesicles isolated from seminal plasma upon Assisted Reproductive Technology (oocyte maturation, fertilization and embryo culture) in a mammalian model)
Periodo di rendicontazione: 2021-03-01 al 2023-02-28
In vitro embryo production (IVEP) is an assisted reproductive technology (ART) with an enormous potential in the biomedical and agricultural fields. In humans, IVEP is the most effective treatment to overcome infertility. In animals, IVEP is an important reality in cattle industry, and it is considered an essential tool for basic research, accelerate genetic progress in animal production and as model for human medicine. However, the IVEP-efficiency remains still relatively low.
In recent years, there have been growing research interest in extracellular vesicles (EVs), nanoparticles (30-to-1000 nm) released by most functional cells into the extracellular environment. Based on their size and biogenetic pathways, they are categorized into exosomes (small EVs) and microvesicles (large EVs). One of the greatest drawbacks that limits the EV-research field is the lack of standardization on the isolation of EVs and their subtypes. An accurate isolation of EV-subtypes is essential for better understating of the role played by each EV-subtype in physio- and pathological processes (including reproductive) and for its potential use as biomarkers.
The EVs have been postulated as crucial messengers for modulating gamete/embryo–maternal interactions in the maternal tract. Studies conducted in livestock species have reported that EVs isolated from female reproductive fluids improved IVEP efficiency. However, no study has addressed which EV-subtype could be involved in these beneficial effects. Seminal plasma (SP), a fluid composed by secretions from male accessory sex glands, play a key role for sperm-genital tract interaction. SP contains a heterogenous population of EVs, which modulate sperm physiological processes. The hypothesis of SPARTEVs was that SP-EVs could play a pivotal role in the successful of IVEP, tackling the problematic of IVEP efficiency from a novel point of view.
The main objectives of SPARTEVs were to (1) enhance the current knowledge of SP-EV subtypes, and (2) to evaluate whether these EVs may improve the efficiency of IVEP. The main conclusions achieved were that: (1) SP-EV-subtypes can be accurately isolated using a size-exclusion chromatography based-method according to its size (large and small); (2) SP-EV subtypes can be taken up by the cumulus cells of oocytes and by capacitated sperm, but not by the oocyte/presumptive zygote; (3) supplementation with SP-EV subtypes: (a) during oocyte in vitro maturation (IVM), modify the gene expression of cumulus cells but do not affect oocyte maturation rates and (b) during in vitro fertilization (IVF), impairs sperm-oocyte binding leading to a decrease on IVF-rates, modulating sperm function; (4) SP-EV subtypes differs in their proteomic profile, suggesting different biogenesis and biological function and source; (5) SP-EVs contains immunoregulatory molecules (transforming growth factor (TGF) -β1, -β2 and 3), which may be involved on modulating uterine immune environment.
In recent years, there have been growing research interest in extracellular vesicles (EVs), nanoparticles (30-to-1000 nm) released by most functional cells into the extracellular environment. Based on their size and biogenetic pathways, they are categorized into exosomes (small EVs) and microvesicles (large EVs). One of the greatest drawbacks that limits the EV-research field is the lack of standardization on the isolation of EVs and their subtypes. An accurate isolation of EV-subtypes is essential for better understating of the role played by each EV-subtype in physio- and pathological processes (including reproductive) and for its potential use as biomarkers.
The EVs have been postulated as crucial messengers for modulating gamete/embryo–maternal interactions in the maternal tract. Studies conducted in livestock species have reported that EVs isolated from female reproductive fluids improved IVEP efficiency. However, no study has addressed which EV-subtype could be involved in these beneficial effects. Seminal plasma (SP), a fluid composed by secretions from male accessory sex glands, play a key role for sperm-genital tract interaction. SP contains a heterogenous population of EVs, which modulate sperm physiological processes. The hypothesis of SPARTEVs was that SP-EVs could play a pivotal role in the successful of IVEP, tackling the problematic of IVEP efficiency from a novel point of view.
The main objectives of SPARTEVs were to (1) enhance the current knowledge of SP-EV subtypes, and (2) to evaluate whether these EVs may improve the efficiency of IVEP. The main conclusions achieved were that: (1) SP-EV-subtypes can be accurately isolated using a size-exclusion chromatography based-method according to its size (large and small); (2) SP-EV subtypes can be taken up by the cumulus cells of oocytes and by capacitated sperm, but not by the oocyte/presumptive zygote; (3) supplementation with SP-EV subtypes: (a) during oocyte in vitro maturation (IVM), modify the gene expression of cumulus cells but do not affect oocyte maturation rates and (b) during in vitro fertilization (IVF), impairs sperm-oocyte binding leading to a decrease on IVF-rates, modulating sperm function; (4) SP-EV subtypes differs in their proteomic profile, suggesting different biogenesis and biological function and source; (5) SP-EVs contains immunoregulatory molecules (transforming growth factor (TGF) -β1, -β2 and 3), which may be involved on modulating uterine immune environment.
The first work was to establish a procedure to isolate EV-subtypes (small and large) from porcine SP. This method was developed combining differential centrifugation, size-exclusion chromatography, and ultrafiltration, allowing the accurate isolation of both EVs subtypes. The second work was to evaluate the putative uptake of SP-EV subtypes by oocyte/sperm/presumptive zygote. The results evidenced that both SP-EV subtypes were taken up by cumulus cells and sperm, but not by the oocyte and presumptive zygote. The third and fourth works were aimed to evaluate whether supplementing IVM and IVF medium with both SP-EV subtypes improved IVM and IVF rates. In IVM, both SP-EV subtypes were able to modify gene expression in cumulus cells (particularly steroidogenesis-related genes), but no effect on oocyte nuclear maturation and on hormone levels in spent medium was observed. In IVF, both SP-EV subtypes decreased sperm-oocyte binding, impairing IVF rates, exerting an effect on sperm metabolism. The fifth task was to decipher the proteomic profile of both SP-EV subtypes. A total of 1,034 proteins were identified and 737 of them were quantified, revealing quantitative proteomic differences among SP-EV subtypes. An additional task was to assess whether SP-EVs could serve as carrier of immunoregulatory molecules, particularly TGF-βs. The results reported that SP-EVs would be involved in secretion of the three TGF-β isoforms and in their transport from the male to the female reproductive tract.
The results of SPARTEVs project have been presented at international conferences (24th Annual Conference of the European Society for Domestic Animal Reproduction, 19th International Congress on Animal Reproduction – ICAR 2020+2, 16th International Congress of the Spanish Society for Animal Reproduction, 10th and 11th International Society for Extracellular Vesicles Conference) and published JCR-journals. In addition, the results have been shared with general public, through social media (Twitter, LinkedIn), by a public webpage and in social events (“Game of research 2022”; “MSCA Seminar at DIMEVET”).
The results of SPARTEVs project have been presented at international conferences (24th Annual Conference of the European Society for Domestic Animal Reproduction, 19th International Congress on Animal Reproduction – ICAR 2020+2, 16th International Congress of the Spanish Society for Animal Reproduction, 10th and 11th International Society for Extracellular Vesicles Conference) and published JCR-journals. In addition, the results have been shared with general public, through social media (Twitter, LinkedIn), by a public webpage and in social events (“Game of research 2022”; “MSCA Seminar at DIMEVET”).
This project was conducted in pig, considered as a suitable animal model for basic, biotechnological as well as biomedical research purposes. SPARTEVs has led to establish a procedure capable to accurately isolate EV subtypes from pig SP, serving as a basis for the isolation of SP-EV subtypes from SP of other species and in other body fluids. SPARTEVs has also evidenced that both SP-EV-subtypes were able to be up taken by cumulus cells when they were added to IVM of oocytes, modifying their gene expression, without affecting oocyte nuclear maturation rates. These results evidenced that SP-EVs-subtypes can interact with female gamete, opening the possibility of a new area of research. Finally, this project also reported, that the supplementation with both pig SP-EV subtypes during IVF, decreases the IVF rates modulating sperm metabolism. This finding would indicate that SP-EVs would exert a direct effect on sperm, impairing sperm-oocyte binding, that may condition fertilization success. SPARTEVs has also deciphered the proteome profile of both pig SP-EV-subtypes. This achievement will allow a better understanding of the specific role played by each SP-EV subtype in reproductive processes and serve as steppingstone in the identification of biomarkers. This project evidenced, for the first time, that SP-EVs would act as carriers of immunoregulatory molecules (TGF β1, β2 and β3) indicating their key role in modulating the immune response of female genital tract, essential for successful embryo development. This finding may should be as basis for further studies in other mammalian species, including humans, considering that these SP-EV-immunoregulatory molecules may hold the key to some pathological processes (e.g. infertility), or even, serve as useful tool to improve ART outcomes. Overall, this project has addressed two H2020-challenges “Health, Demographic Change and Wellbeing” and “Food security; sustainable agriculture and forestry, marine and inland water research and the bioeconomy”.