Pré-maturação in vitro de oócitos bovinos com NPPC e vesículs extracelulares obtidas em diferentes fases da onda de crescimento folicular
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2022-02-17
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Universidade Estadual Paulista (Unesp)
Resumo
Oócitos bovinos submetidos à maturação in vitro (MIV) retomam espontaneamente a maturação nuclear, no entanto, este padrão não é observado em relação às maturações citoplasmática e molecular. Desta forma, tais oócitos constituem uma população heterogênea em relação aos eventos que compõem a maturação oocitária para aquisição de competência. Este estudo propôs avaliar os efeitos de um sistema de pré-maturação in vitro (préMIV) de complexos cumulus oócitos (CCOs) bovinos com o uso do bloqueador precursor do peptídeo natriurético tipo C (NPPC) e com vesículas extracelulares (VEs) obtidas de folículos em diferentes fases do desenvolvimento [pré-desvio ~ 7 mm (PreDev); desvio ~8,5 mm (DevF1); pós-desvio ~12 mm (PostDev) e; pré-ovulatório > 12 mm (PreOv)], sobre: (1) a captação das VEs pelas células dos CCOs; (2) a transferência de RNA das VEs para os oócitos; (3) o padrão de compactação da cromatina e aquisição de competência oocitária para o desenvolvimento embrionário e; (4) parâmetros de qualidade dos embriões provenientes deste sistema. Ao final do período de 8 horas do sistema de préMIV foi possível evidenciar, em todos os grupos tratados com VEs (PreDev, DevF1, PostDev e PreOv), a captura das mesmas pelas células dos CCOs, bem como a transferência de RNA proveniente destas vesículas para o ooplasma, sendo que os oócitos do grupo DevF1 apresentaram concentração de RNA aumentada no ooplasma, quando comparados aos oócitos dos demais grupos (P<0.05). Em relação ao padrão de compactação da cromatina (vesícula germinativa – VG – de 0 a 3) dos CCOs, independente do grupo avaliado (Imaturo – imediatamente após a remoção do folículo ovariano, Controle – meio de préMIV sem adição de VEs, PreDev, DevF1, PostDev e PreOv), não houve diferença (P>0.05) quanto às categorias VG0 e VG2; já as categorias de VG1e VG3 diferiram entre os grupos (P<0.05), sendo que o grupo Imaturo apresentou maior taxa de oócitos em VG1 (61,9%), em relação a todos os grupos, e menor taxa de oócitos em VG3 (5,7%) quando comparado aos grupos PreDev (45,4) e PostDev (48,1%) (P<0.05). O cultivo de préMIV associado ou não às VEs não promoveu aumento das taxas de clivagem (Controle 82%, PreDev 81,5%, DevF1 75%, PostDev 76% e PreOv 76,9%) (P>0.05) e desenvolvimento embrionário (Controle 43,6%, PreDev 38,6%, DevF1 48,1%, PostDev 38,3% e PreOv 40,4%) (P>0.05). Em relação aos parâmetros de qualidade embrionária, o grupo PostDev apresentou maior número de blastômeros em relação ao grupo Controle (P<0.05), porém as avaliações de potencial de membrana mitocondrial e porcentagem de células apoptóticas não diferiram, independente do tratamento (P>0.05). Concluímos, portanto, através dos achados na literatura, que o trabalho aqui apresentado é o primeiro a utilizar um sistema de pré-maturação in vitro de complexos cumulus oócitos bovinos enriquecido com vesículas extracelulares e, evidenciamos, a partir dos resultados que o sistema permitiu a interação entre as células do complexo cumulus oócitos e vesículas extracelulares, independente da fase do desenvolvimento folicular em que as vesículas foram obtidas. Adicionalmente, foi possível evidenciar melhorias na qualidade dos embriões sugerindo efeitos benéficos deste sistema.
Oocytes that undergo in vitro maturation (IVM) spontaneously resume nuclear maturation, however, this pattern is not observerd in cytoplasmic and molecular maturation. This situation creates a heterogeneous oocyte population which influences the developmental competence acquisition. For this reason, the present study proposed to evaluate the effects of an in vitro prematuration (preIVM) system of bovine cumulus oocyte complex (COC) with C-type natriuretic peptide precursor (NPPC) and extracelular vesicles (EVs) from follicles at different stages of development [pre-deviation ~ 7 mm (PreDev); deviation ~ 8,5 mm (DevF1); post-deviation ~ 12 mm (PostDev) and; pre-ovulatory > 12 mm (PreOv)], on: (1) uptake of EVs by COC cells; (2) RNA transfer from EVs to oocytes; (3) COC chromatin configuration and developmental competence acquisition; (4) embryos quality parameters. After 8 hours of preIVM, it was possible to evidence, in all groups treated with EVs (PreDev, DevF1, PostDev and PreOv), their capture by COCs, as well as, the transfer of RNA from these vesicles to the ooplasm. The DevF1 group was the only group, when compared to the other groups, that showed an increase in the cytoplasmatic RNA concentration (P<0.05). The assessment of COC chromatin configuration (germinal vesicle – GV – 0 to 3), regardless of the group (Immature – after follicle immediately removal, Control – preIVM medium withour EVs, PreDev, DevF1, PostDev and PreOv) no difference (P>0.5) was noted for GV0 and GV2 categories. Whereas the GV1 and GV3 categories differed between groups (P>0.05), the group with the highest percentage of oocytes in GV1 was the Immature group (61.9%), this group also had a lower rate of oocytes in GV3 (5.7%) when compared to the PreDev (45,4%) and PostDev (48.1%) groups (P<0.05). The preIVM culture, with ou without EVs, did not promote an increase in cleavage rates (D3) (Control 82%, PreDev 81.5%, DevF1 75%, PostDev 76% and PreOv 76.9%) (P>0.05) and embryonic development rates (D7) (Control 43.6%, PreDev 38.6%, DevF1 48.1%, PostDev 38.3% and PreOv 40.4%) (P>0.05). Embryos from PostDev group had more blastomere numbers than the control group (P<0.05). However, the assessments of mitochondrial membrane potential and apoptotic cells did not differ independent of treatment (P<0.05). In conclusion, this is the first study in the literature to use an in vitro pre-maturation system of bovine cumulus oocyte complex enriched with extracelular vesicles and through the results presented here we confirm that the in vitro pre-maturation system of bovine cumulus oocyte complex allowed the interaction between the cumulus cells and extracelular vesicles regardless of the stage of folicular development in wich the vesicles were obtained. Additionally, it was possible to evidence improvements in the quality of the embryos suggestiong beneficial effects of this system.
Oocytes that undergo in vitro maturation (IVM) spontaneously resume nuclear maturation, however, this pattern is not observerd in cytoplasmic and molecular maturation. This situation creates a heterogeneous oocyte population which influences the developmental competence acquisition. For this reason, the present study proposed to evaluate the effects of an in vitro prematuration (preIVM) system of bovine cumulus oocyte complex (COC) with C-type natriuretic peptide precursor (NPPC) and extracelular vesicles (EVs) from follicles at different stages of development [pre-deviation ~ 7 mm (PreDev); deviation ~ 8,5 mm (DevF1); post-deviation ~ 12 mm (PostDev) and; pre-ovulatory > 12 mm (PreOv)], on: (1) uptake of EVs by COC cells; (2) RNA transfer from EVs to oocytes; (3) COC chromatin configuration and developmental competence acquisition; (4) embryos quality parameters. After 8 hours of preIVM, it was possible to evidence, in all groups treated with EVs (PreDev, DevF1, PostDev and PreOv), their capture by COCs, as well as, the transfer of RNA from these vesicles to the ooplasm. The DevF1 group was the only group, when compared to the other groups, that showed an increase in the cytoplasmatic RNA concentration (P<0.05). The assessment of COC chromatin configuration (germinal vesicle – GV – 0 to 3), regardless of the group (Immature – after follicle immediately removal, Control – preIVM medium withour EVs, PreDev, DevF1, PostDev and PreOv) no difference (P>0.5) was noted for GV0 and GV2 categories. Whereas the GV1 and GV3 categories differed between groups (P>0.05), the group with the highest percentage of oocytes in GV1 was the Immature group (61.9%), this group also had a lower rate of oocytes in GV3 (5.7%) when compared to the PreDev (45,4%) and PostDev (48.1%) groups (P<0.05). The preIVM culture, with ou without EVs, did not promote an increase in cleavage rates (D3) (Control 82%, PreDev 81.5%, DevF1 75%, PostDev 76% and PreOv 76.9%) (P>0.05) and embryonic development rates (D7) (Control 43.6%, PreDev 38.6%, DevF1 48.1%, PostDev 38.3% and PreOv 40.4%) (P>0.05). Embryos from PostDev group had more blastomere numbers than the control group (P<0.05). However, the assessments of mitochondrial membrane potential and apoptotic cells did not differ independent of treatment (P<0.05). In conclusion, this is the first study in the literature to use an in vitro pre-maturation system of bovine cumulus oocyte complex enriched with extracelular vesicles and through the results presented here we confirm that the in vitro pre-maturation system of bovine cumulus oocyte complex allowed the interaction between the cumulus cells and extracelular vesicles regardless of the stage of folicular development in wich the vesicles were obtained. Additionally, it was possible to evidence improvements in the quality of the embryos suggestiong beneficial effects of this system.
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Maturação in vitro de oócitos, Bovino, Reprodução, Tecnologias reprodutivas