Publication: Interaction of the ejaculate and extracellular vesicles of the porcine female reproductive tract: biotechnological implications on fertility, embryo development, and offspring
Authors
Toledo Guardiola, Santa María
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Escuela Internacional de Doctorado
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Matas Parra, Carmen ; Soriano Úbeda, Cristina de las Mercedes
Publisher
Universidad de Murcia
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DOI
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info:eu-repo/semantics/doctoralThesis
Description
Tesis por compendio de publicaciones
Abstract
Esta tesis doctoral se presenta como un compendio de cinco publicaciones científicas que contribuyen a una línea de investigación centrada en el análisis del impacto de las vesículas extracelulares (EVs) derivadas de los fluidos reproductivos en los gametos, la fecundación, la formación del embrión y la generación de una descendencia viable. El objetivo de esta tesis fue el de, través de un enfoque multidimensional, analizar este impacto evaluando el efecto paterno en parámetros hematológicos de la descendencia, la angiogénesis uterina, la caracterización de las EVs en los fluidos oviductal y uterino y la regulación de éstas en la funcionalidad espermática, la fecundación in vitro y el consiguiente desarrollo embrionario.
El Capítulo 1 estableció una base de estudio a través de una revisión bibliográfica que remarcó y justificó los experimentos llevados a cabo durante esta tesis doctoral.
El resto de los capítulos se llevaron a cabo siguiendo tres diseños experimentales diferentes: En primer lugar, se inseminaron cerdas con dosis homospérmicas de verracos fértiles y se analizaron parámetros hematológicos de la descendencia. Por otro lado, se realizó inseminación artificial (AI) con distintas fracciones del eyaculado de verraco en cerdas prolíficas, se recolectaron los tractos genitales de éstas y se analizaron cuerpos lúteos, embriones y tejido uterino a los seis días post-inseminación. Además, se examinó el contenido proteico de las EVs del fluido oviductal (oEVs) y uterino (uEVs) obtenidos en estos tractos genitales y se aisló y analizó por proteómica su contenido. Por último, se aislaron y caracterizaron oEVs porcinas (poEVs) de cerdas de matadero en diferentes fases del ciclo estral y se evaluó su efecto sobre el espermatozoide mediante ensayos de motilidad, viabilidad, estado acrosómico, fosforilación proteica y fecundación in vitro.
El Capítulo 2 identificó una correlación estadística entre las características específicas del semen de los verracos, particularmente aquellas asociadas con el daño al ADN espermático y los parámetros de salud en lechones. También se observó que aquellos verracos que mostraban mejor calidad seminal generaron camadas con mayor resistencia a la hipoglucemia y mejor crecimiento postnatal. En conclusión, la calidad espermática del verraco tiene un impacto profundo tanto en la fertilidad, como en la salud y viabilidad de la descendencia.
El Capítulo 3 determinó que la inseminación con diferentes fracciones del eyaculado afectó significativamente al desarrollo embrionario, con un mayor porcentaje de mórulas en el grupo inseminado con la fracción rica en espermatozoides (SRF). Concluyendo que, las fracciones del eyaculado modulan la respuesta uterina y el desarrollo embrionario.
El Capítulo 4 encontró que el contenido proteico de las uEVs y oEVs dependía de la fracción del eyaculado utilizada y se identificaron proteínas clave relacionadas con la fertilidad como EZR, HSPAA901 y PDS. En resumen, se observó que las EVs secretadas por el tracto reproductor femenino tenían un rol activo en la modulación del proteoma uterino y la funcionalidad espermática.
Finalmente, el Capítulo 5 notificó que las poEVs de la fase folicular tardía mostraron una mayor afinidad por los espermatozoides, afectando a su motilidad, capacitación y a la tasa de penetración espermática, disminuyendo la polispermia en cerdo. Esto, nos llevó a la conclusión de que las poEVs secretadas durante las distintas fases del ciclo estral interactúan con los espermatozoides, alterando su comportamiento y su capacidad fecundante.
Estos hallazgos ahondan en el conocimiento de los mecanismos de comunicación intercelular que se dan durante el proceso reproductivo, tanto en las etapas de pre- como post- fecundación. Además, este conocimiento podría servir de base para el desarrollo de la producción animal, como el perfeccionamiento de la selección de reproductores y de estrategias de AI, así como para el avance de sectores como la industria biomédica
This doctoral thesis is presented as a compendium of five scientific publications that contribute to a line of research focused on the analysis of the impact of the extracellular vesicles (EVs) derived from reproductive fluids on gametes, fertilization, embryo formation and the generation of viable offspring. The objective of this thesis was to analyze this impact using a multidimensional approach, evaluating the paternal effect on offspring's hematological parameters that can be predicted by the boar sperm parameters, uterine angiogenesis in response to the ejaculate fractions presence, EVs characterization in oviductal and uterine fluid, and EVs regulation of sperm functionality, in vitro fertility and ulterior embryo development. Chapter 1 establishes the basis of the study through a bibliographic review that remarks and justifies the experimental research carried out in this doctoral thesis. The rest of the chapters were conducted using three different experimental designs: Initially, sows were inseminated with homospermic doses of highly fertile boars, and the hematological parameters of the offspring were subsequently analyzed. Conversely, artificial insemination (AI) was performed with different fractions of boar ejaculate in sows. Six days post-AI, the genital tracts were collected, and corpora lutea, embryos and uterine tissue were analyzed. Furthermore, EVs from oviductal (oEVs) and uterine fluid (uEVs) were isolated from these genital tracts, and the protein content of these vesicles was analyzed by proteomics. Finally, porcine oEVs (poEVs) from slaughterhouse sows at different stages of the estrous cycle were isolated and characterized, and their effect on sperm was evaluated using motility, viability, acrosomal status, protein phosphorylation, and in vitro fertilization assays. Chapter 2 identified a statistic correlation between specific boar semen characteristics, particularly those associated with sperm DNA damage, and critical parameters of piglet health, including markers of organ function, metabolism, and stress resistance. It was also observed that boars with superior semen quality produced litters that exhibit indicators of potential resistance to hypoglycemia and enhanced postnatal growth. In conclusion, it was proved that the quality of the boar's sperm exerts a substantial influence on both fertility and viability and health of the offspring. Chapter 3 determined that insemination with different ejaculate fractions significantly affected embryo development, with a higher percentage of morulae observed in the sperm rich fraction (SRF) inseminated group. Furthermore, it was observed that uterine vascularization depended on the ejaculate fraction used. Therefore, it was ascertained that distinct ejaculate fractions modulate uterine response and embryonic development. Chapter 4 found that the protein content of uEVs and oEVs depends on the ejaculate fraction utilized. The identification of key proteins associated with reproductive processes was undertaken, with particular attention to proteins related to fertility, including EZR, HSPAA901, and PDS. In summary, it was observed that EVs secreted by the female reproductive tract have an active role in modulating the uterine proteome and sperm functionality. Finally, Chapter 5 addressed that late follicular phase poEVs exhibited an augmented propensity for binding to sperm, thereby exerting a substantial influence on their motility and capacitation. Furthermore, it was observed that poEVs exerted a detrimental influence on sperm penetration rates, leading to a reduction in polyspermy in pigs. This led us to the conclusion that poEVs secreted during different phases of the estrous cycle interact with sperm, modifying their behavior and fertilization capacity. These findings contribute to our understanding of the mechanisms of intercellular communication during the reproductive process, both in the pre- and post-fertilization stages. Moreover, this knowledge could serve as a foundation for developments related to animal production, such as the refinement of breeding stock selection and artificial insemination strategies, as well as advancements in sectors such as the biomedical industry.
This doctoral thesis is presented as a compendium of five scientific publications that contribute to a line of research focused on the analysis of the impact of the extracellular vesicles (EVs) derived from reproductive fluids on gametes, fertilization, embryo formation and the generation of viable offspring. The objective of this thesis was to analyze this impact using a multidimensional approach, evaluating the paternal effect on offspring's hematological parameters that can be predicted by the boar sperm parameters, uterine angiogenesis in response to the ejaculate fractions presence, EVs characterization in oviductal and uterine fluid, and EVs regulation of sperm functionality, in vitro fertility and ulterior embryo development. Chapter 1 establishes the basis of the study through a bibliographic review that remarks and justifies the experimental research carried out in this doctoral thesis. The rest of the chapters were conducted using three different experimental designs: Initially, sows were inseminated with homospermic doses of highly fertile boars, and the hematological parameters of the offspring were subsequently analyzed. Conversely, artificial insemination (AI) was performed with different fractions of boar ejaculate in sows. Six days post-AI, the genital tracts were collected, and corpora lutea, embryos and uterine tissue were analyzed. Furthermore, EVs from oviductal (oEVs) and uterine fluid (uEVs) were isolated from these genital tracts, and the protein content of these vesicles was analyzed by proteomics. Finally, porcine oEVs (poEVs) from slaughterhouse sows at different stages of the estrous cycle were isolated and characterized, and their effect on sperm was evaluated using motility, viability, acrosomal status, protein phosphorylation, and in vitro fertilization assays. Chapter 2 identified a statistic correlation between specific boar semen characteristics, particularly those associated with sperm DNA damage, and critical parameters of piglet health, including markers of organ function, metabolism, and stress resistance. It was also observed that boars with superior semen quality produced litters that exhibit indicators of potential resistance to hypoglycemia and enhanced postnatal growth. In conclusion, it was proved that the quality of the boar's sperm exerts a substantial influence on both fertility and viability and health of the offspring. Chapter 3 determined that insemination with different ejaculate fractions significantly affected embryo development, with a higher percentage of morulae observed in the sperm rich fraction (SRF) inseminated group. Furthermore, it was observed that uterine vascularization depended on the ejaculate fraction used. Therefore, it was ascertained that distinct ejaculate fractions modulate uterine response and embryonic development. Chapter 4 found that the protein content of uEVs and oEVs depends on the ejaculate fraction utilized. The identification of key proteins associated with reproductive processes was undertaken, with particular attention to proteins related to fertility, including EZR, HSPAA901, and PDS. In summary, it was observed that EVs secreted by the female reproductive tract have an active role in modulating the uterine proteome and sperm functionality. Finally, Chapter 5 addressed that late follicular phase poEVs exhibited an augmented propensity for binding to sperm, thereby exerting a substantial influence on their motility and capacitation. Furthermore, it was observed that poEVs exerted a detrimental influence on sperm penetration rates, leading to a reduction in polyspermy in pigs. This led us to the conclusion that poEVs secreted during different phases of the estrous cycle interact with sperm, modifying their behavior and fertilization capacity. These findings contribute to our understanding of the mechanisms of intercellular communication during the reproductive process, both in the pre- and post-fertilization stages. Moreover, this knowledge could serve as a foundation for developments related to animal production, such as the refinement of breeding stock selection and artificial insemination strategies, as well as advancements in sectors such as the biomedical industry.
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