Maduración de ovocitos y desarrollo de embriones bovinos cocultivados en monocapas celulares
During the twentieth century, the introduction of innovative reproductive\nbiotechnologies in cattle production has allowed a breakthrough in this field. The embryo transfer produced by in vitro fertilization (IVF) increased significantly and\nin 2013 reached a global record high of 42% of transferr...
Guardado en:
| Autor principal: | |
|---|---|
| Otros Autores: | |
| Formato: | Tesis doctoral acceptedVersion |
| Lenguaje: | Español |
| Publicado: |
Facultad de Ciencias Veterinarias
2018
|
| Materias: | |
| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_3060 http://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_3060.dir/3060.PDF |
| Aporte de: |
| id |
I28-R145-HWA_3060 |
|---|---|
| record_format |
dspace |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-145 |
| collection |
Repositorio Digital de la Universidad de Buenos Aires (UBA) |
| language |
Español |
| orig_language_str_mv |
spa |
| topic |
Cocultivo Células de la granulosa bovina Células luteales bovinas Producción in vitro de embriones bovinos Ovocito Maduración Vaca Desarrollo embrionario Células Monocultivos Reproducción |
| spellingShingle |
Cocultivo Células de la granulosa bovina Células luteales bovinas Producción in vitro de embriones bovinos Ovocito Maduración Vaca Desarrollo embrionario Células Monocultivos Reproducción Maruri, Alejandro Maduración de ovocitos y desarrollo de embriones bovinos cocultivados en monocapas celulares |
| topic_facet |
Cocultivo Células de la granulosa bovina Células luteales bovinas Producción in vitro de embriones bovinos Ovocito Maduración Vaca Desarrollo embrionario Células Monocultivos Reproducción |
| description |
During the twentieth century, the introduction of innovative reproductive\nbiotechnologies in cattle production has allowed a breakthrough in this field. The embryo transfer produced by in vitro fertilization (IVF) increased significantly and\nin 2013 reached a global record high of 42% of transferred embryos. However, despite scientific efforts to optimize systems for in vitro production (IVP), the culture conditions remain suboptimal. Thus, the performance and quality of\nembryos are lower than those produced in vivo thereby generating higher early embryonic losses that negatively affect production.\nCoculture with somatic cells could improve suboptimal conditions and\npromote embryonic development through the production of mitogenic compounds\nand substances that promote cell differentiation. Also, through detoxifying and modifying embryotoxins concentrations and energy substrates. Besides, the\nstudy of coculture systems would provide valuable information about the\nrequirements for early embryonic development, biochemistry of somatic cells and their interaction with the embryo. The aim of this thesis is to optimize the bovine embryo production from IVF, using systems of coculture with granulosa and luteal cells from bovine, to\nincrease yield and/or quality of blastocysts produced. The effect of two coculture\nsystems with bovine granulosa cells: granulosa cells line 1 (BGC-1) and\ngranulosa cells from passage 1 after primary culture (CGB-1), on in vitro\nmaturation were assessed. Similarly, unpurified cells from a primary culture of\nbovine corpus luteum were used but the results were inconclusive. Furthermore,\nthe effect of a coculture system with purified bovine luteal cells of passage 1\n(CLB-1) during early embryonic development was evaluated. The presence of\nBGC-1 showed no beneficial effect on oocyte nuclear maturation compared to\nthe control group (without cells). Varying maturation rates were observed, and in\nmost cases, meiotic resumption was inhibited. By employing an alternative model with CGB-1 significantly increases the nuclear maturation rates (85%) compared\nto the control (70%). However, there were no differences in Annexin V-FITC rate (determination for early apoptosis) or dead rate in oocyte between groups. Also, late apoptosis (TUNEL assay) was evaluated and there were no differences\nbetween groups (CGB-1: 1.12% vs. control 1.33%). After IVM, oocyte levels of\nreactive oxygen species (ROS) were determined using DCHFDA assay and it\nshowed a significant increase in coculture group with CGB-1. As an indirect\nassessment parameter of oocyte cytoplasmic maturation, IVF assays were\nperformed. In this case, the control group received the gonadotropins. The\ncleavage rate was determined at 48 h post-IVF and was similar in both groups (CGB-1: 80.5% vs. control: 79.4%). However, blastocysts rate was significantly lower in control than the coculture (CGB-1: 14% vs. control: 25.6%).\nThe embryo coculture with CLB-1 was performed during the first 48 h of in\nvitro culture (IVC). The CLB were previously isolated and purified by differential\ncentrifugation on a discontinuous Percoll? density gradient, and the purity was determined by immunocytochemistry (ICC) using antibodies against 3?-HSD (enzyme that catalyzes progesterone biosynthesis from pregnenolone). All the\ncells selected (fractions 5 and 6 of the Percoll? gradient) were positive to 3?HSD. Nile red dye was used to detect cytoplasmic lipid droplets and all the cells\nassessed were positive indicating steroidogenic activity preserved. On the other hand, cells from passage 1 without selection showed 80.6% ± 7,02 of positive cells. This coculture system modified the kinetic of embryo development respect to a conventional system that not uses cells (control). A significantly increase in blastocyst rate (CLB-1: 50% vs. control: 30%) and stage 6-blastocyst rate (CLB1: 37% vs. control: 24%) was observed. The ROS level was measured in day 2- embryos and was significantly higher in the coculture. Furthermore, the detection of the proliferation antigen Ki-67, as an early marker of embryo quality, was proposed. The cell proliferation rate and the mean number of blastomeres were\ndetermined in day 2-embryos using immunofluorescence for Ki-67 and Hoechst\n33342 staining. The cell proliferation rate in embryos cultured with CLB-1 was\nsignificantly higher than the control but there were no differences in a mean\nnumber of blastomeres. Late apoptosis (TUNEL assay) was assessed in blastocysts and the rate of apoptotic blastomeres was lower in coculture with CLB-1 than the control (4.10% vs. 10.90%). In conclusion, the coculture system with BGC-1 is not an alternative to\noptimize PIV of the bovine embryo. On the other hand, the coculture with CGB-1\nshowed a positive effect on nuclear maturation but their use was not superior\nregarding efficiency about traditional PIV system. The effects of this coculture\nwere evident delayed, and the lower yield may be associated with an increased level of ROS in oocytes. Finally, the use of CLB-1 in coculture during early embryo\ndevelopment showed a significant embryotrophic effect with high blastocyst yield of a better quality, thereby optimizing the PIV of the bovine embryo.\n |
| author2 |
Lombardo, Daniel M. |
| author_facet |
Lombardo, Daniel M. Maruri, Alejandro |
| format |
Tesis doctoral Tesis doctoral acceptedVersion |
| author |
Maruri, Alejandro |
| author_sort |
Maruri, Alejandro |
| title |
Maduración de ovocitos y desarrollo de embriones bovinos cocultivados en monocapas celulares |
| title_short |
Maduración de ovocitos y desarrollo de embriones bovinos cocultivados en monocapas celulares |
| title_full |
Maduración de ovocitos y desarrollo de embriones bovinos cocultivados en monocapas celulares |
| title_fullStr |
Maduración de ovocitos y desarrollo de embriones bovinos cocultivados en monocapas celulares |
| title_full_unstemmed |
Maduración de ovocitos y desarrollo de embriones bovinos cocultivados en monocapas celulares |
| title_sort |
maduración de ovocitos y desarrollo de embriones bovinos cocultivados en monocapas celulares |
| publisher |
Facultad de Ciencias Veterinarias |
| publishDate |
2018 |
| url |
http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_3060 http://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_3060.dir/3060.PDF |
| work_keys_str_mv |
AT marurialejandro maduraciondeovocitosydesarrollodeembrionesbovinoscocultivadosenmonocapascelulares |
| _version_ |
1766017524920483840 |
| spelling |
I28-R145-HWA_30602020-02-21 During the twentieth century, the introduction of innovative reproductive\nbiotechnologies in cattle production has allowed a breakthrough in this field. The embryo transfer produced by in vitro fertilization (IVF) increased significantly and\nin 2013 reached a global record high of 42% of transferred embryos. However, despite scientific efforts to optimize systems for in vitro production (IVP), the culture conditions remain suboptimal. Thus, the performance and quality of\nembryos are lower than those produced in vivo thereby generating higher early embryonic losses that negatively affect production.\nCoculture with somatic cells could improve suboptimal conditions and\npromote embryonic development through the production of mitogenic compounds\nand substances that promote cell differentiation. Also, through detoxifying and modifying embryotoxins concentrations and energy substrates. Besides, the\nstudy of coculture systems would provide valuable information about the\nrequirements for early embryonic development, biochemistry of somatic cells and their interaction with the embryo. The aim of this thesis is to optimize the bovine embryo production from IVF, using systems of coculture with granulosa and luteal cells from bovine, to\nincrease yield and/or quality of blastocysts produced. The effect of two coculture\nsystems with bovine granulosa cells: granulosa cells line 1 (BGC-1) and\ngranulosa cells from passage 1 after primary culture (CGB-1), on in vitro\nmaturation were assessed. Similarly, unpurified cells from a primary culture of\nbovine corpus luteum were used but the results were inconclusive. Furthermore,\nthe effect of a coculture system with purified bovine luteal cells of passage 1\n(CLB-1) during early embryonic development was evaluated. The presence of\nBGC-1 showed no beneficial effect on oocyte nuclear maturation compared to\nthe control group (without cells). Varying maturation rates were observed, and in\nmost cases, meiotic resumption was inhibited. By employing an alternative model with CGB-1 significantly increases the nuclear maturation rates (85%) compared\nto the control (70%). However, there were no differences in Annexin V-FITC rate (determination for early apoptosis) or dead rate in oocyte between groups. Also, late apoptosis (TUNEL assay) was evaluated and there were no differences\nbetween groups (CGB-1: 1.12% vs. control 1.33%). After IVM, oocyte levels of\nreactive oxygen species (ROS) were determined using DCHFDA assay and it\nshowed a significant increase in coculture group with CGB-1. As an indirect\nassessment parameter of oocyte cytoplasmic maturation, IVF assays were\nperformed. In this case, the control group received the gonadotropins. The\ncleavage rate was determined at 48 h post-IVF and was similar in both groups (CGB-1: 80.5% vs. control: 79.4%). However, blastocysts rate was significantly lower in control than the coculture (CGB-1: 14% vs. control: 25.6%).\nThe embryo coculture with CLB-1 was performed during the first 48 h of in\nvitro culture (IVC). The CLB were previously isolated and purified by differential\ncentrifugation on a discontinuous Percoll? density gradient, and the purity was determined by immunocytochemistry (ICC) using antibodies against 3?-HSD (enzyme that catalyzes progesterone biosynthesis from pregnenolone). All the\ncells selected (fractions 5 and 6 of the Percoll? gradient) were positive to 3?HSD. Nile red dye was used to detect cytoplasmic lipid droplets and all the cells\nassessed were positive indicating steroidogenic activity preserved. On the other hand, cells from passage 1 without selection showed 80.6% ± 7,02 of positive cells. This coculture system modified the kinetic of embryo development respect to a conventional system that not uses cells (control). A significantly increase in blastocyst rate (CLB-1: 50% vs. control: 30%) and stage 6-blastocyst rate (CLB1: 37% vs. control: 24%) was observed. The ROS level was measured in day 2- embryos and was significantly higher in the coculture. Furthermore, the detection of the proliferation antigen Ki-67, as an early marker of embryo quality, was proposed. The cell proliferation rate and the mean number of blastomeres were\ndetermined in day 2-embryos using immunofluorescence for Ki-67 and Hoechst\n33342 staining. The cell proliferation rate in embryos cultured with CLB-1 was\nsignificantly higher than the control but there were no differences in a mean\nnumber of blastomeres. Late apoptosis (TUNEL assay) was assessed in blastocysts and the rate of apoptotic blastomeres was lower in coculture with CLB-1 than the control (4.10% vs. 10.90%). In conclusion, the coculture system with BGC-1 is not an alternative to\noptimize PIV of the bovine embryo. On the other hand, the coculture with CGB-1\nshowed a positive effect on nuclear maturation but their use was not superior\nregarding efficiency about traditional PIV system. The effects of this coculture\nwere evident delayed, and the lower yield may be associated with an increased level of ROS in oocytes. Finally, the use of CLB-1 in coculture during early embryo\ndevelopment showed a significant embryotrophic effect with high blastocyst yield of a better quality, thereby optimizing the PIV of the bovine embryo.\n Fil: Maruri, Alejandro. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Buenos Aires. Argentina Facultad de Ciencias Veterinarias Lombardo, Daniel M. Maruri, Alejandro 2018-12-27 Durante el siglo XX, la introducción de biotecnologías reproductivas\ninnovadoras en la producción bovina le ha permitido a la ganadería un gran avance. La transferencia de embriones producidos por fecundación in vitro (FIV) aumentó significativamente, alcanzando en el año 2013 un máximo histórico a\nnivel mundial de 42% del total de embriones transferidos. Sin embargo, a pesar de los esfuerzos científicos para optimizar los sistemas de producción in vitro (PIV), las condiciones de cultivo continúan siendo subóptimas. En consecuencia,\nel rendimiento y la calidad de los embriones es inferior respecto a los producidos\nin vivo lo que genera mayores pérdidas embrionarias, afectando negativamente\nla producción.\nEl cocultivo con células somáticas podría mejorar las condiciones\nsubóptimas y favorecer el desarrollo embrionario a través de la producción de\ncompuestos con actividad mitogénica y sustancias que promueven la\ndiferenciación celular, la detoxificación de embriotoxinas y la modificación de las\nconcentraciones de sustratos energéticos. Asimismo, el estudio de sistemas de cocultivo podría proporcionar información valiosa sobre los requerimientos para\nel desarrollo embrionario temprano, la bioquímica de las células somáticas y su\ninteracción con el embrión.\nEl objetivo general de la tesis es optimizar la producción de embriones\nbovinos obtenidos por FIV, a través de la utilización de sistemas de cocultivo con\ncélulas de granulosa y luteales bovinas, para incrementar el rendimiento y/o la calidad de los blastocistos obtenidos a través de dicha biotecnología. Se evaluó\nel efecto de dos sistemas de cocultivo con células de granulosa bovina durante\nla maduración in vitro (MIV): células de la línea BGC-1 (bovine granulosa cell line\n1) y células del primer pasaje luego del cultivo primario (CGB-1). Paralelamente,\nse utilizaron células de pasaje 1 de cultivo primario de cuerpo lúteo bovino sin\npurificar, no obteniéndose resultados concluyentes. De igual modo, se evaluó el\nefecto de un sistema de cocultivo con células luteales bovinas purificadas de pasaje 1 (CLB-1) durante el desarrollo embrionario temprano. La presencia de\nBGC-1 no demostró tener un efecto beneficioso sobre la maduración nuclear \novocitaria respecto al grupo control (sin células). Se observaron porcentajes\nvariables y en la mayoría de los casos la reanudación meiótica fue inhibida. Al\nemplear como modelo alternativo las CGB-1, se incrementó significativamente el\nporcentaje de maduración nuclear (85%) respecto al control (70%). Sin embargo,\nlos porcentajes de ovocitos Anexina V-FITC positivos (determinación para\napoptosis temprana) y de ovocitos muertos luego de la MIV no mostraron\ndiferencias entre el cocultivo con CGB-1 y el control. También, se evaluó la\napoptosis tardía (ensayo TUNEL) sin observar diferencias entre los grupos\n(CGB-1: 1,12% vs. control: 1,33%). Los niveles ovocitarios de especies reactivas derivadas del oxígeno (ERO) se determinaron a través del ensayo de DCHFDA\nluego de la MIV, observando un incremento significativo de los mismos en el\ncocultivo con CGB-1. Como parámetro indirecto de evaluación de la maduración\ncitoplasmática se realizaron experimentos de FIV, en cuyo caso se agregaron\ngonadotrofinas al medio de MIV en el grupo control. El porcentaje de embriones\nsegmentados se determinó a las 48 h post-FIV y fue similar en ambos grupos\n(CGB-1: 80,5% vs. control: 79,4%). Sin embargo, el rendimiento de blastocistos\nfue significativamente menor en el cocultivo (CGB-1: 14% vs. control: 25,6%).\nEl cocultivo de embriones con CLB-1 se realizó durante las primeras 48 h\nde cultivo in vitro (CIV). Las CLB se aislaron y purificaron previamente mediante\ncentrifugación diferencial en un gradiente discontinuo de Percoll®, determinando\nla pureza mediante inmunocitoquímica (ICQ) para 3?-HSD (enzima que cataliza la biosíntesis de progesterona a partir de la pregnenolona). Se observó que la totalidad de las células de pasaje 1 que habían sido seleccionadas (fracciones 5\ny 6 del gradiente de Percoll®) fueron positivas a 3?-HSD. Utilizando el colorante rojo Nilo se determinó la presencia de vacuolas lipídicas citoplasmáticas en la\ntotalidad de las células evaluadas, lo cual indicó actividad esteroidogénica\nconservada. Del mismo modo, las células de pasaje 1 sin selección mostraron\nun porcentaje del 80,6% ± 7,02 de positividad al colorante. El cocultivo con este sistema modificó la cinética de desarrollo embrionario respecto al sistema convencional sin células (control). Se observó un incremento significativo del porcentaje de blastocistos (CLB-1: 50% vs. control: 30%) y de blastocistos de\nestadio 6 (CLB-1: 37% vs. control: 24%). Los niveles de ERO se determinaron\nen embriones de día 2, siendo significativamente superiores en el cocultivo. Por otro lado, se planteó la detección del factor de proliferación celular Ki-67 como marcador temprano de calidad embrionaria. Mediante inmunofluorescencia para\nKi-67 y tinción con Hoechst 33342 se determinó el porcentaje de proliferación\ncelular y el número promedio de blastómeras en embriones de día 2. El\nporcentaje de proliferación celular en los embriones cocultivados con CLB-1 fue\nsignificativamente mayor respecto al control sin existir diferencias en el número\nde blastómeras. Se evaluó la apoptosis tardía (ensayo TUNEL) en blastocistos,\nobservando un menor porcentaje de blastómeras apoptóticas en el cocultivo con CLB-1 respecto al control (4,10% vs. 10,90%).\nEn conclusión, el sistema de cocultivo con BGC-1 no es una alternativa\npara optimizar la PIV de embriones bovinos. Sin embargo, el cocultivo con CGB1 mostró un efecto positivo sobre la maduración nuclear ovocitaria, no obstante, su utilización no resultó ser superior en términos de eficiencia respecto al sistema\nde PIV tradicional. Los efectos de este sistema de cocultivo se evidenciaron\ntardíamente y los menores rendimientos podrían asociarse a los mayores niveles de ERO en los ovocitos. Finalmente, la utilización de CLB-1 purificadas y en\ncocultivo durante las etapas tempranas de desarrollo demostró un marcado\nefecto embriotrófico con altos porcentajes de blastocistos de mejor calidad, optimizando la PIV de embriones bovinos. application/pdf Cocultivo Células de la granulosa bovina Células luteales bovinas Producción in vitro de embriones bovinos spa Universidad de Buenos Aires. Facultad de Ciencias Veterinarias info:eu-repo/semantics/openAccess https://creativecommons.org/licenses/by-ncnd/2.5/ar/ Ovocito Maduración Vaca Desarrollo embrionario Células Monocultivos Reproducción Maduración de ovocitos y desarrollo de embriones bovinos cocultivados en monocapas celulares info:eu-repo/semantics/doctoralThesis info:ar-repo/semantics/tesis doctoral info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_3060 http://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_3060.dir/3060.PDF |