Efeitos da associação do sistema pré-MIV com NPPC com MIV suplementada de modo sequencial com LH sobre a produção in vitro de embriões bovinos
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Data
2019-02-28
Autores
Pronunciate, Micheli
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Universidade Estadual Paulista (Unesp)
Resumo
In vivo, a maturação oocitária é gonadotrofina-dependente (FSH e LH) e, até haver o devido estímulo, o oócito é mantido em parada meiótica (prófase I) devido à manutenção dos níveis intraoocitários de cAMP. O FSH é o responsável pelo crescimento inicial do folículo antral enquanto o LH promove a maturação final oocitária (no estágio pós-desvio), sinalizando a produção de peptídeos EGF-like (fator de crescimento semelhante ao epidermal) nas células da granulosa. Estes se ligam ao seu receptor (EGFR) nas células do cumulus ativando a via da proteína cinase ativadora de mitógeno (MAPK), causando o fechamento das junções comunicantes do tipo GAP e culminando na retomada meiótica. Os peptídeos AREG (Ampirregulina)e EREG (Epirregulina) são utilizados na maturação in vitro em diferentes espécies e em substituição ou associação com o FSH. Alguns estudos demonstram que, quando maturados com esses peptídeos, os oócitos desenvolvem embriões de melhor qualidade, com maior número de células da massa celular interna e mais parecidos com aqueles maturados in vivo. No entanto, o oócito retirado do microambiente folicular retoma a meiose espontaneamente, de forma precoce e não sincronizada entre o núcleo e o citoplasma. Para melhorar os resultados da maturação in vitro, é importante retardar a retomada espontânea meiótica. A literatura descreve a eficácia do precursor do peptídeo natriurético tipo C (NPPC) em manter in vitro a concentração de cAMP oocitária similar à fisiológica. Diante disso, neste estudo foi observado o efeito do AREG sobre a abundância de transcritos alvo de oócitos e células do cumulus, além do seu efeito em adição às gonadotrofinas (LH ou FSH) quanto à progressão meiótica e a produção de embriões, quando precedida pela pré-MIV com NPPC. Nossos resultados corroboram que o AREG é eficiente tanto quanto o FSH na maturação de complexos cumulus-oócito e não altera as taxas de quebra da vesícula germinativa (GVBD) ou oócitos que chegaram a meiose II (MII). No entanto, o perfil dos transcritos alvo estudado apresentou diferença entre ambos, indicando distintas ações celulares do AREG e do FSH. Demonstramos ainda que o LH, sozinho, é ineficiente na maturação e produziu uma alta taxa de GV mesmo após 24 horas. No entanto, quando a maturação foi precedida por pré-MIV utilizando NPPC, a taxa de blastocisto e a abundância dos transcritos alvo, o LH não diferiu dos demais grupos. Em suma, como já descrito na literatura, o AREG foi eficaz para a MIV e não estimulou a expressão de receptores de LH nas células do cumulus. Adicionalmente, o uso do NPPC na pré-MIV foi capaz de retardar a retomada da meiose, por tempo suficiente para que o oócito adquirisse competência para suportar o desenvolvimento embrionário inicial.
In vivo, the oocyte maturation is gonadotropin-dependent (LH and FSH) and, until there is a due stimulation, the oocyte is maintained in meiotic arrest (prophase I) due to the maintenance of intra-oocyte levels of cAMP. The FSH is responsible for initial growth of the antral follicle while the LH promotes final oocyte maturation (post-deviation), signaling the production of EGF-like (Epidermal Growth Factor-like) peptides in the granulosa cells. They bind to their receptor (EGFR) on cumulus cells activating the mitogen-activating protein kinase (MAPK) pathway causing closure of Gap Junctions culminating in meiosis resumption. The AREG (amphiregulin) and EREG (epiregulin) peptides are used in vitro maturation in different species and in substitution or association with FSH. Same studies shown that when mature with these peptide, oocytes develop better-quality embryos, with more cells in the inner cell mass, and more similar to those in vivo. However, the oocyte removed from the follicular microenvironment, resumes meiosis spontaneously, early and not synchronized between the cytoplasm and nucleus. To improve the in vitro maturation results, it is important delay spontaneous meiotic resumption. The literature describes the efficacy of the C-type natriuretic peptide (NPPC) precursor in maintaining the physiological-like oocyte cAMP concentration in vitro. In this study, the effect of AREG on the abundance of oocyte and cumulus cell transcript was observed, as well as its effect in addition to gonadotrophins (LH or FSH) on meiotic progression and embryo production, when preceded by pre-IVM with NPPC. Our results corroborate that the AREG is efficient as well as FSH in maturation of cumulus-oocyte complex (COC) and does not alter the rates of germinal vesicle breakdown (GVBD) or oocytes that reached meiosis II (MII) However, the target transcript profile showed a difference between the two, indicating distinct cellular actions of the AREG and FSH. We also demonstrate that LH, alone, is inefficient in maturation and produced a high GV rate even after 24 hours. However, when the maturation was preceded by pre-IVM using NPPC, blastocyst rate and abundance of target transcripts in the LH group did not differ from the other groups. In summary as describe in the literature, AREG was effective for IVM and did not stimulate the expression of LH receptor in cumulus cells. In addition, the use of NPPC in pre-IVM can postpone the meiosis resumption, long enough for the oocyte to acquire competence to support early embryonic development.
In vivo, the oocyte maturation is gonadotropin-dependent (LH and FSH) and, until there is a due stimulation, the oocyte is maintained in meiotic arrest (prophase I) due to the maintenance of intra-oocyte levels of cAMP. The FSH is responsible for initial growth of the antral follicle while the LH promotes final oocyte maturation (post-deviation), signaling the production of EGF-like (Epidermal Growth Factor-like) peptides in the granulosa cells. They bind to their receptor (EGFR) on cumulus cells activating the mitogen-activating protein kinase (MAPK) pathway causing closure of Gap Junctions culminating in meiosis resumption. The AREG (amphiregulin) and EREG (epiregulin) peptides are used in vitro maturation in different species and in substitution or association with FSH. Same studies shown that when mature with these peptide, oocytes develop better-quality embryos, with more cells in the inner cell mass, and more similar to those in vivo. However, the oocyte removed from the follicular microenvironment, resumes meiosis spontaneously, early and not synchronized between the cytoplasm and nucleus. To improve the in vitro maturation results, it is important delay spontaneous meiotic resumption. The literature describes the efficacy of the C-type natriuretic peptide (NPPC) precursor in maintaining the physiological-like oocyte cAMP concentration in vitro. In this study, the effect of AREG on the abundance of oocyte and cumulus cell transcript was observed, as well as its effect in addition to gonadotrophins (LH or FSH) on meiotic progression and embryo production, when preceded by pre-IVM with NPPC. Our results corroborate that the AREG is efficient as well as FSH in maturation of cumulus-oocyte complex (COC) and does not alter the rates of germinal vesicle breakdown (GVBD) or oocytes that reached meiosis II (MII) However, the target transcript profile showed a difference between the two, indicating distinct cellular actions of the AREG and FSH. We also demonstrate that LH, alone, is inefficient in maturation and produced a high GV rate even after 24 hours. However, when the maturation was preceded by pre-IVM using NPPC, blastocyst rate and abundance of target transcripts in the LH group did not differ from the other groups. In summary as describe in the literature, AREG was effective for IVM and did not stimulate the expression of LH receptor in cumulus cells. In addition, the use of NPPC in pre-IVM can postpone the meiosis resumption, long enough for the oocyte to acquire competence to support early embryonic development.
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Palavras-chave
Oócito, Células do cumulus, AREG, NPPC