Identificación de vesículas extracelulares y su carga proteica, secretadas por los túbulos de almacenamiento espermático del oviducto de hembras de pavo criollo (Meleagris gallopavo), y su efecto sobre espermatozoides conservados in vitro.
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Date
2024
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Universidad de Concepción
Abstract
Las hembras aviares, como las pavas, poseen la particularidad de presentar una “fertilización sostenida” en el tiempo, dependiente de la capacidad de la hembra para almacenar y liberar esperma en el transcurso de los ciclos ovulatorios, capacidad que es conferida por estructuras especializadas, llamadas “túbulos de almacenamiento espermático” (TAE), localizadas en la unión útero vaginal (UUV). Los TAE permiten asegurar la viabilidad de los espermatozoides tras la conservación por períodos prolongados de hasta 70 días en pavas. Sin embargo, a pesar del gran interés práctico que tendría para su conservación in vitro, las condiciones bioquímicas, mecanismos fisiológicos y la base genética que permiten la conservación y supervivencia de los espermatozoides aviares son prácticamente desconocidas. El líquido oviductal ha sido indicado como el principal responsable para que espermatozoides presenten un entorno óptimo durante los eventos reproductivos, identificándose a las vesículas extracelulares (EVs) como su principal constituyente. Las EVs son definidas como mediadores en la comunicación intercelular mediante el transporte de múltiples componentes moleculares bioactivos desde la célula de origen hacia la célula receptora, transportando proteínas, lípidos, ARN y ADN. De éste modo se postula que el contenido de las EVs de la UUV, donde están los TAE, del oviducto de hembras de pavo criollo (Meleagris gallopavo), inhibe la movilidad sin afectar la viabilidad espermática. Frente a lo cual se buscó establecer la relación entre el fotoperiodo estacional y las temporadas reproductivas, para determinar la concentración y contenido proteico de las EVs procedentes de la UUV del oviducto de hembras de pavo criollo, en diversas condiciones estacionales y reproductivas, probando la influencia de las EVs sobre la movilidad y viabilidad espermática in vitro. Para ello se efectuaron análisis de perfiles séricos de hormonas sexuales en hembras (estrógeno y progesterona) y machos (testosterona), mediante la técnica de radioinmunoensayo (RIA), a partir de lo cual se determinaron las temporadas de muestreo, de acuerdo al fotoperiodo y estado ovulatorio de las hembras. Se extrajeron EVs, mediante la técnica de ultracentrifugación, a partir del fluido oviductal (in vivo) y el sobrenadante de cultivos celulares (in vitro) de las regiones oviductales del M (sin TAE) y la UUV (con TAE), de acuerdo a las etapas reproductivas de fotoperiodo negativo (FN), fotoperiodo positivo (FP) y FP en estado ovulatorio (FP en O). Se efectuó una caracterización biométrica por medio de las técnicas de NTA y TEM, y se efectuó una caracterización molecular mediante espectometría de masas. Una vez determinadas las muestras de EVs con mejores resultados bioactivos, correspondiente a las muestras in vivo de M y la UUV en FP en O, se efectuaron pruebas de interacción, movilidad y viabilidad espermática mediante la co-incubación de EVs con espermatozoides, a partir de tres concentraciones de EVs (4 x 107, 4 x 108 y 4 x 1010 partículas/mL) y 4 puntos en el tiempo (2, 4, 6 y 10 horas de co-incubación). Empleando las técnicas de visualización con PKH para interacción, microscopía para evaluación de movilidad, y la tinción de eosina y visualización por microscopía para viabilidad. Los resultados permitieron señalar que los picos de hormonas sexuales se desarrollan entre fines de invierno e inicios de primavera, correspondiendo al período de mayor interés reproductivo para la especie. Se pudo establecer que el tamaño de las EVs in vivo (en FN, FP y FP en O) dependiente del estado ovulatorio y es independiente del fotoperiodo y el tejido, mientras que la concentración es dependiente del fotoperiodo y el tejido. Respecto al tamaño de las EVs de muestras in vivo e in vitro (en FP con y sin O), es dependiente del estado ovulatorio y el origen de la muestra, mientras que la concentración sólo depende del origen de la muestra. En cuanto al contenido proteico de las EVs, depende principalmente del origen de las muestras, validándose el método de lavado oviductal (in vivo) por sobre el método in vitro, observándose un diferencial de expresión (ANXA5, STX2, LZTFL1, STXBP1, ATP2B4, RAB34, APOB, TTLL3) respecto a las funciones biológicas de movilidad espermática y reacción acrosomal. En relación a la coincubación de EVs de la UUV con espermatozoides, se pudo establecer la internalización a partir de las 2 hrs, con conservación de la viabilidad y reducción de la movilidad en función del tiempo. De éste modo se puede establecer que existen ciclos reproductivos definidos de acuerdo al fotoperiodo estacional, con EVs secretadas en función del estado reproductivo, origen de la muestra y tejido oviductal, con mayor fidelidad de la realidad secretora a partir de muestras de lavado oviductal, con EVs que transportan proteínas importantes para la función espermática, colaborando positivamente en su viabilidad y movilidad.
Avian females, like turkeys hen (Meleagris gallopavo), have the particularity of presenting “sustained fertilization” over time, dependent on the female's ability to store and release sperm during the ovulatory cycles, a capacity that is conferred by specialized structures., called “sperm storage tubules” (SST), located in the utero-vaginal junction (UVJ). SSTs ensure the viability of sperm after conservation for prolonged periods of up to 70 days in turkeys hen. owever, despite the great practical interest that it would have for its in vitro conservation, the biochemical conditions, physiological mechanisms and genetic basis that allow the conservation and survival of avian spermatozoa are practically unknown. The oviductal fluid has been indicated as the main responsible for spermatozoa presenting an optimal environment during reproductive events, identifying extracellular vesicles (EVs) as its main constituent. EVs are defined as mediators in intercellular communication through the transport of multiple bioactive molecular components from the cell of origin to the recipient cell, transporting proteins, lipids, RNA and DNA. In this way, it is postulated that the content of the EVs of the UVJ, where the SSTs are located, of the oviduct of turkeys hen, inhibits motility without affecting sperm viability. Against which we sought to establish the relationship between the seasonal photoperiod and the reproductive seasons, to determine the concentration and protein content of the EVs from the UVJ of the oviduct of female Creole turkeys, in various seasonal and reproductive conditions, testing the influence of EVs on sperm motility and viability in vitro. For this, analyzes of serum profiles of sexual hormones were carried out in females (estrogen and progesterone) and males (testosterone), using the radioimmunoassay technique, from which the sampling seasons were determined, according to the photoperiod and ovulatory state of females. EVs were extracted, using the ultracentrifugation technique, from the oviductal fluid (in vivo) and the supernatant of cell cultures (in vitro) from the oviductal regions of the M (without SST) and the UVJ (with SST), according to the reproductive stages of negative photoperiod (NP), positive photoperiod (PP) and PP in the ovulatory state (PP in O). A biometric characterization was carried out using NTA and TEM techniques, and a molecular characterization was carried out using mass spectrometry. Once the EVs samples with the best bioactive results were determined, corresponding to the in vivo samples of M and UVJ in PP in O, interaction, motility and sperm viability tests were carried out by co-incubating EVs with sperm, from of three concentrations of EVs (4 x 107, 4 x 108 and 4 x 1010 particles/mL) and 4 points in time (2, 4, 6 and 10 hours of co-incubation). Using visualization techniques with PKH for interaction, microscopy for mobility assessment, and eosin staining and visualization by microscopy for viability. The results allowed us to point out that the peaks of sexual hormones develop between the end of winter and the beginning of spring, corresponding to the period of greatest reproductive interest for the species. It was possible to establish that the size of EVs in vivo (in NP, PP and PP in O) depends on the ovulatory state and is independent of the photoperiod and tissue, while the concentration is dependent on the photoperiod and tissue. Regarding the size of EVs from in vivo and in vitro samples (in PP with and without O), it depends on the ovulatory state and the origin of the sample, while the concentration only depends on the origin of the sample. Regarding the protein content of EVs, it depends mainly on the origin of the samples, validating the oviductal washing method (in vivo) over the in vitro method, observing a differential expression (ANXA5, STX2, LZTFL1, STXBP1, ATP2B4, RAB34, APOB, TTLL3) regarding the biological functions of sperm motility and acrosomal reaction. In relation to the co incubation of UVJ EVs with sperm, internalization could be established after 2 hours, with conservation of viability and reduction of motility as a function of time. In this way it can be established that there are reproductive cycles defined according to the seasonal photoperiod, with EVs secreted depending on the reproductive state, origin of the sample and oviductal tissue, with greater fidelity of the secretory reality from oviductal washing samples, with EVs that transport proteins important for sperm function, positively collaborating in their viability and mobility.
Avian females, like turkeys hen (Meleagris gallopavo), have the particularity of presenting “sustained fertilization” over time, dependent on the female's ability to store and release sperm during the ovulatory cycles, a capacity that is conferred by specialized structures., called “sperm storage tubules” (SST), located in the utero-vaginal junction (UVJ). SSTs ensure the viability of sperm after conservation for prolonged periods of up to 70 days in turkeys hen. owever, despite the great practical interest that it would have for its in vitro conservation, the biochemical conditions, physiological mechanisms and genetic basis that allow the conservation and survival of avian spermatozoa are practically unknown. The oviductal fluid has been indicated as the main responsible for spermatozoa presenting an optimal environment during reproductive events, identifying extracellular vesicles (EVs) as its main constituent. EVs are defined as mediators in intercellular communication through the transport of multiple bioactive molecular components from the cell of origin to the recipient cell, transporting proteins, lipids, RNA and DNA. In this way, it is postulated that the content of the EVs of the UVJ, where the SSTs are located, of the oviduct of turkeys hen, inhibits motility without affecting sperm viability. Against which we sought to establish the relationship between the seasonal photoperiod and the reproductive seasons, to determine the concentration and protein content of the EVs from the UVJ of the oviduct of female Creole turkeys, in various seasonal and reproductive conditions, testing the influence of EVs on sperm motility and viability in vitro. For this, analyzes of serum profiles of sexual hormones were carried out in females (estrogen and progesterone) and males (testosterone), using the radioimmunoassay technique, from which the sampling seasons were determined, according to the photoperiod and ovulatory state of females. EVs were extracted, using the ultracentrifugation technique, from the oviductal fluid (in vivo) and the supernatant of cell cultures (in vitro) from the oviductal regions of the M (without SST) and the UVJ (with SST), according to the reproductive stages of negative photoperiod (NP), positive photoperiod (PP) and PP in the ovulatory state (PP in O). A biometric characterization was carried out using NTA and TEM techniques, and a molecular characterization was carried out using mass spectrometry. Once the EVs samples with the best bioactive results were determined, corresponding to the in vivo samples of M and UVJ in PP in O, interaction, motility and sperm viability tests were carried out by co-incubating EVs with sperm, from of three concentrations of EVs (4 x 107, 4 x 108 and 4 x 1010 particles/mL) and 4 points in time (2, 4, 6 and 10 hours of co-incubation). Using visualization techniques with PKH for interaction, microscopy for mobility assessment, and eosin staining and visualization by microscopy for viability. The results allowed us to point out that the peaks of sexual hormones develop between the end of winter and the beginning of spring, corresponding to the period of greatest reproductive interest for the species. It was possible to establish that the size of EVs in vivo (in NP, PP and PP in O) depends on the ovulatory state and is independent of the photoperiod and tissue, while the concentration is dependent on the photoperiod and tissue. Regarding the size of EVs from in vivo and in vitro samples (in PP with and without O), it depends on the ovulatory state and the origin of the sample, while the concentration only depends on the origin of the sample. Regarding the protein content of EVs, it depends mainly on the origin of the samples, validating the oviductal washing method (in vivo) over the in vitro method, observing a differential expression (ANXA5, STX2, LZTFL1, STXBP1, ATP2B4, RAB34, APOB, TTLL3) regarding the biological functions of sperm motility and acrosomal reaction. In relation to the co incubation of UVJ EVs with sperm, internalization could be established after 2 hours, with conservation of viability and reduction of motility as a function of time. In this way it can be established that there are reproductive cycles defined according to the seasonal photoperiod, with EVs secreted depending on the reproductive state, origin of the sample and oviductal tissue, with greater fidelity of the secretory reality from oviductal washing samples, with EVs that transport proteins important for sperm function, positively collaborating in their viability and mobility.
Description
Tesis presentada para optar al grado de Doctora en Ciencias Veterinarias
Keywords
Pavos - Genética, Espermatozoides - Análisis, Vesículas extracelulares