Nível de transcrição relativa de BMP15, BAX e CASP3 determinado pelo qRT-PCR em complexos cumulus-oócitos imaturos vitrificados de equinos
DOI:
https://doi.org/10.5433/1679-0359.2022v43n3p927Palavras-chave:
Proteína X associada a Bcl-2, Proteína morfogenética óssea 15, Expressão genética, Caspase 3, Equinos.Resumo
Pesquisas sobre a vitrificação de gametas femininos estão sendo realizadas para o desenvolvimento de um método confiável de criopreservação dos complexos cumulus-oócitos (CCOs) na espécie equina. Apesar da implementação intensiva de biotecnologias reprodutivas em equinos, a vitrificação dos CCOs imaturos permanece em estágio experimental em relação à competência celular. O objetivo do estudo foi determinar o nível de transcrição relativo dos genes Proteína morfogenética óssea 15 (BMP15); Proteína X associada a Bcl-2 (BAX); e Caspase 3 (CASP3) em CCOs equinos antes e após a vitrificação. Folículos ovarianos foram aspirados de ovários coletados em matadouro. O total de 240 CCOs foi coletado e distribuído em grupos vitrificados (VIT, n=120) e não vitrificados (N-VIT, n=120). Os CCOs foram preservados e as expressões transcritas relativas de BMP15, BAX, CASP3 foram determinadas pela técnica de qRT-PCR sendo normalizadas em relação ao GAPDH. Além disso, 38 CCOs foram avaliados para determinar a configuração da cromatina no estágio de vesícula germinativa antes e após a vitrificação pela exposição a 10 ug/ml de bisbenzimida. Os resultados mostraram uma diferença no nível de abundância de mRNA dos CCOs para o gene BAX entre os grupos VIT (2,05 ± 0,47) e N-VIT (0,85 ± 0,08). Não houve diferença no nível de transcrição relativa do mRNA de CASP3 e BMP15 nos CCOs do grupo N-VIT (0,63 ± 0,20 e 1,55 ± 0,73, respectivamente) em comparação com VIT (0,64 ± 0,01 e 2,84 ± 2,20, respectivamente). Todos os CCOs foram considerados em estágio imaturo de desenvolvimento, embora os CCOs no grupo N-VIT apresentaram a configuração de cromatina condensada em maior número de células avaliadas em comparação com VIT (100% vs 60,7%, respectivamente). Demonstramos que BMP15 e CASP3 são detectados em CCOs imaturos em VIT e N-VIT. Conclui-se que o BAX é altamente expresso em CCOs equinos imaturos vitrificados sendo relacionado à sinalização de apoptose em células expostas ao processo de vitrificação.Downloads
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Referências
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Choi, Y., & Hinrichs, K. (2017). Vitrification of in vitro-produced and in vivo-recovered equine blastocysts in a clinical program. Theriogenology, 87, 48-54. doi: 10.1016/j.theriogenology.2016.08.005
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Curcio, B., Gastal, M., Pereira, G., Corcini, C., Landim-Alvarenga, F., Barros, S.,... Gastal, E. (2014). Ultrastructural morphology and nuclear maturation rates of immature equine oocytes vitrified with different solutions and exposure times. Journal of Equine Veterinary Science, 34(5), 632-640. doi: 10.10 16/j.jevs.2013.12.002
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De Coster, T., Velez, D. A., Van Soom, A., Woelders, H., & Smits, K. (2020). Cryopreservation of equine oocytes: looking into the crystal ball. Reproduction Fertility and Development, 32(5), 453-467. doi: 10. 1071/RD19229
Ducheyne, K. D., Rizzo, M., Daels, P. F., Stout, T. A. E., & de Ruijter-Villani, M. (2019). Vitrifying immature equine oocytes impairs their ability to correctly align the chromosomes on the MII spindle. Reproduction Fertility and Development, 31(8), 1330-1338. doi: 10.1071/RD18276
Ebrahimi, B., Valojerdi, M., Eftekhari-Yazdi, P., Baharvand, H., & Farrokhi, A. (2010). IVM and gene expression of sheep cumulus-oocyte complexes following different methods of vitrification. Reproductive BioMedicine Online, 20(1), 26-34. doi: 10.1016/j.rbmo.2009.10.020
Eldridge-Panuska, W., Caracciolo di Brienza, V., Seidel, G., Squires, E., & Carnevale, E. (2005). Establishment of pregnancies after serial dilution or direct transfer by vitrified equine embryos. Theriogenology, 63(5), 1308-1319. doi: 10.1016/j.theriogenology.2004.06.015
Galli, C., Colleoni, S., Duchi, R., Lagutina, I., & Lazzari, G. (2013). Equine assisted reproduction and embryo technologies. Animal Reproduction, 10(3), 334-343. Retrieved from https://www.animal-reproduction. org/article/5b5a604cf7783717068b46a1/pdf/animreprod-10-3-334.pdf
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Hinrichs, K. (2018). Assisted reproductive techniques in mares. Reproduction in Domestic Animals, 53(2), 4-13. doi: 10.1111/rda.13259
Hinrichs, K., & Schmidt, A. (2000). Meiotic competence in horse oocytes: interactions among chromatin configuration, follicle size, cumulus morphology, and season. Biology of Reproduction, 62(5), 1402-1408. doi: 10.1095/biolreprod62.5.1402
Hinrichs, K., Choi, Y., Love, L., Varner, D., Love, C., & Walckenaer, B. (2005). Chromatin configuration within the germinal vesicle of horse oocytes: changes post mortem and relationship to meiotic and developmental competence. Biology of Reproduction, 72(5), 1142-1150. doi: 10.1095/biolreprod.104.03 6012
Hinrichs, K., Schmidt, A., Friedman, P., Selgarth, J., & Martin, M. (1993). In vitro maturation of horse oocytes: characterization of chromatin configuration using fluorescence microscopy. Biology of Reproduction, 48(2), 363-370. doi: 10.1095/biolreprod48.2.363
Hurtt, A., Landim-Alvarenga, F., Seidel, G., & Squires, E. (2000). Vitrification of immature and mature equine and bovine oocytes in an ethylene glycol, ficoll and sucrose solution using open-pulled straws. Theriogenology, 54(1), 119-128. doi: 10.1016/S0093-691X(00)00330-7
Hussein, T., Froiland, D., Amato, F., Thompson, J., & Gilchrist, R. (2005). Oocytes prevent cumulus cell apoptosis by maintaining a morphogenic paracrine gradient of bone morphogenetic proteins. Journal of Cell Science, 118(22), 5257-5268. doi: 10.1242/jcs.02644
Jang, W., Lee, S., Choi, H., Lim, J., Heo, Y., Cui, X., & Kim, N. (2014). Vitrification of immature mouse oocytes by the modified-cut standard straw method. Cell Biology International, 38(2), 164-171. doi: 10. 1002/cbin.10163
Kim, M., & Tilly, J. (2004). Current concepts in Bcl-2 family member regulation of female germ cell development and survival. Biochim Biophys Acta, 1644(2-3), 205-220. doi: 10.1016/j.bbamcr.2003.10. 012
Knight, P., & Glister, C. (2003). Local roles of TGF-b superfamily members in the control of ovarian follicle development. Animal Reproduction Science, 78(3-4), 165-183. doi: 10.1016/S0378-4320(03)00089-7
Lan, Z., Gu, P., Xu, X., Jackson, K., Demayo, F., O’Malley, B., & Cooney, A. (2003). GCNF-dependent repression of BMP-15 and GDF-9 mediates gamete regulation of female fertility. EMBO Journal, 22(1), 4070-4081. doi: 10.1093/emboj/cdg405
Leibo, S. P. (2008). Cryopreservation of oocytes and embryos: optimization by theoretical versus empirical analysis. Theriogenology, 69(1), 37-47. doi: 10.1016/j.theriogenology.2007.10.006
Leon, P., Campos, V., Kaefer, C., Begnini, K., McBride, A., Dellagostin, O.,… Collares, T. (2013). Expression of apoptotic genes in immature and in vitro matured equine oocytes and cumulus cells. Zygote, 21(3), 279-285. doi: 10.1017/S0967199411000554
Li, Y., Li, R., Ou, S., Zhang, N., Ren, L., Wei, L.,… Yang, D. (2014). Increased GDF9 and BMP15 mRNA levels in cumulus granulosa cells correlate with oocyte maturation, fertilization, and embryo quality in humans. Reproduction Biology and Endocrinology, 12, 81. doi: 10.1186/1477-7827-12-81
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2022-02-28
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Polenz, M. F., Pereira, G. R., Rissi, V. B., Bertolin, K., Gonçalves, P. B., Augusto, G. C., & Rechsteiner, S. M. F. (2022). Nível de transcrição relativa de BMP15, BAX e CASP3 determinado pelo qRT-PCR em complexos cumulus-oócitos imaturos vitrificados de equinos. Semina: Ciências Agrárias, 43(3), 927–942. https://doi.org/10.5433/1679-0359.2022v43n3p927
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