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Improving developmental competence of in vitro produced goat embryos
In vitro production
In vitro development
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|摘要:||提昇山羊體外生產胚之品質攸關生殖技術未來之發展與應用。本研究以取自屠宰場卵巢之卵丘卵母細胞複合體（Cumulus-oocyte complexes，COCs），經體外成熟、受精、培養，探討不同胚培養系統對山羊卵母細胞成熟與後續發育潛能之影響。試驗一：以經體外受精後之卵母細胞分別培養於TCM199輸卵管上皮細胞（TCM-GOEC）共培養系統與合成輸卵管液（SOFaa）之培養系統。結果顯示，分裂率在培養於SOFaa培養系統顯著（P < 0.05）高於以TCM-GOEC共培養者，而囊胚率與平均囊胚細胞數在TCM-GOEC共培養組顯著（P < 0.05）高於培養於SOFaa者。試驗二：將COCs分別培養於含不同濃度Shh (Sonic Hedgehog) 之成熟培養液中，成熟後進行受精與體外培養。結果發現Shh的添加（0.25 或 0.5 μg∙mL−1）相較對照組顯著提高卵母細胞之成熟率（92.4%與95.0% 及 86.2%，P < 0.05）。在胚後續發育能力方面，以成熟液中添加0.5 μg∙mL−1之Shh組者相較對照組亦有較高的囊胚率（66.3 ± 10.9% 及 41.4 ± 12.9%，P < 0.05）。經胚移置後，胚胎存活率亦以添加0.5 μg∙mL−1之Shh組者高於對照組（37.5% 及 14.8 %，P < 0.05）。試驗三：探討Shh與其相關蛋白質基因分別在卵巢及卵母細胞上之表現，並進一步分析源自Shh處理之卵母細胞而得之囊胚其基因表現差異。結果顯示，以TCM199輸卵管上皮細胞共培養及體外成熟液中添加Shh皆可改善山羊卵母細胞及其後續發育能力。Shh與其相關受體蛋白於卵巢濾泡皆有表現，且添加Shh於體外成熟培養液中確實促進成熟卵母細胞之Gli1表現。此外，源自Shh處理之卵母細胞發育之囊胚，以基因晶片分析其基因表現，相較於未經Shh處理者，共有185個基因表現差異；再進一步以定量PCR分析基因表現量，顯示calponin-1（CNN1）, pregnancy-associated glycoprotein 1-like （PAG1）, MAP kinase-interacting serine/threonine-protein kinase 2 （MKNK2）及 NYN domain and retroviral integrase containing （NYNRIN）等基因具有顯著較高之表現量。綜上結果，山羊胚體外生產系統利用輸卵管上皮細胞共培養及於體外成熟過程中添加Shh蛋白質可誘導相關基因之表現並提高產出胚之品質與效率。|
Generation of quality in vitro-produced goat embryos is an essential step towards the development and application of reproductive technologies. This study aimed to evaluate the effects of different embryo culture systems on oocyte maturation and subsequent embryonic development in goats. Cumulus-oocyte-complexes (COCs) aspirated from the surface follicles of the ovaries collected from a local abattoir were subjected to in vitro maturation (IVM), fertilization (IVF) and culture. In Exp. 1, the presumptive zygotes were cultured either in TCM-199 with monolayers of goat oviduct epithelial cells (TCM-GOEC) or in synthetic oviduct fluid plus amino acids (SOFaa). Our results showed that the mean cleavage rate of presumptive zygotes cultured in SOFaa was higher (P < 0.05) than that in TCM-GOEC. However, blastocyst rates and the cell numbers per blastocyst in TCM-GOEC were higher (P < 0.05) than those in SOFaa. In Exp. 2, COCs were randomly allocated to the IVM medium supplemented with 0 (Control), 0.125, 0.25, 0.5, or 1.0 μg∙mL−1 recombinant mouse Sonic Hedgehog (Shh) protein. After maturation, COCs were fertilized with frozen-thawed semen and the presumptive zygotes were cultured in TCM-GOEC for 9 days. We found that Shh (0.25 or 0.5 μg∙mL−1) enhanced oocyte maturation as compared to those of the control group (92.4% and 95.0% vs. 86.2%, P < 0.05). An improved blastocyst rate (from 41.4 ± 12.9% to 66.3 ± 10.9%, P < 0.05) was observed in embryos derived from the oocytes treated with 0.5 μg∙mL−1 Shh. After embryo transfer, embryos develpoing to term were higher in the Shh-supplemented group compared to those without Shh supplementation (37.5% vs. 14.8%, P < 0.05). In Exp. 3, follicular oocytes all showed the expression of Shh, Ptched, Smo and Gli1 expressions. During oocytes maturation, Shh supplementation improved (P < 0.05) Gli 1 expression. Blastocysts derived from the oocytes with Shh supplementation in the IVM medium had up- or down-regulated expression of 185 genes. Based on real-time PCR analysis, calponin-1 (CNN1), pregnancy-associated glycoprotein 1-like (PAG1), MAP kinase-interacting serine/threonine-protein kinase 2 (MKNK2) and NYN domain and retroviral integrase containing (NYNRIN) genes were expressed higher in embryos derived from oocytes with Shh treatment. Taken together, the above results reveal that embryo co-cultured with TCM-GOEC and Shh supplementation in the IVM medium can activate some Shh-signaling and expression of its associated genes, which are beneficial for the development of goat oocytes and embryos.
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