- 學位論文
源自不同牛種來源細胞質紡錘體轉置技術之建立及其耐熱性分子機制探討
Establishing the spindle transfer technology to produce cattle with different bovine cytoplasm origins and evaluating the thermotolerance of their ear fibroblasts at the molecular level
摘要
伴隨全球氣候之逐漸暖化，將更加劇熱緊迫之問題，尤其熱緊迫對熱帶-亞熱帶地區酪農業所造成之繁殖內分泌紊亂及衍生之經濟損失亦將更為嚴重。目前荷蘭牛係全球所飼養最多之乳牛品種，屬不耐熱牛種，因此在熱季時將嚴重降低乳產量。所幸，本研究室曾利用核轉置 (somatic cell nuclear transfer, SCNT) 技術證實，即便供核細胞源自不耐熱之荷蘭牛，當卵母細胞源自耐熱之台灣黃牛時，則可改善該胚之耐熱能力。惟SCNT胚將可能遭遇基因再程序化不完全現象，而降低該胚之發育能力；相對的，紡錘體轉置 (spindle transfer, ST) 胚之產製乃將荷蘭牛之細胞核先轉置入台灣黃牛受核卵母細胞內，再以荷蘭牛精子進行受精，由於此等細胞核均為生殖細胞因此不會造成ST胚基因再程序化不完全之現象。惟截至目前，利用細胞質單一精子注射（intracytoplasmic sperm injection, ICSI）或體外受精（in vitro fertilization, IVF）技術用以生產ST胚之技術尚未健全，且仍未有ST牛成功分娩之記錄。基於此，本研究之研究一將首先著重於探討ICSI時機 (ICSI-ST vs. ST-ICSI) 及受精方法 (ICSI vs. IVF) 對ST胚發育能力之影響。結果顯示，利用ICSI-ST流程所產製ST胚之囊胚率（24.7%）顯著 (P < 0.05) 高於利用ST-ICSI流程產製者（5.9%）。此外，ST卵母細胞 (ST-IVF) 較之於卵丘-卵母細胞複合體( cumulus-oocyte complexes, COCs-IVF)、卵丘裸露卵母細胞 (denuded oocytes, DO-IVF)、透明帶穿刺卵母細胞 (zona cutted oocytes, ZC-IVF)等經IVF後，雖具有最高之受精率 (97.3%)，但卻有最高之多精入卵率 (24.7%) 及最低之囊胚率 (22.7%)。惟無論如何，ICSI-ST法 (24.5%)所產製ST胚之囊胚率雖與ST-IVF法 (25.2%)產製者間無顯著差異 (P < 0.05)，但胚移置後之懷孕率 (46.7 vs. 6.3%) 及ST仔畜出生率(33.3 vs. 0.0%) 則均以ST-IVF組顯著高於ICSI-ST組。而本研究所產製5頭ST牛體細胞之細胞質內mtDNA異質型態之比率則介於2~18%之間。然而基於目前有關細胞質源自台灣黃牛細胞核源自荷蘭牛之ST牛 (ST-Yo-Hn) 的耐熱性分子機制探討仍未有研究；因此，於研究二則著重於比較ST-Yo-Hn牛與純種荷蘭牛間耳朵纖維母細胞經熱緊迫 (heat stress, hs) 後相關基因與蛋白質表現差異性分析。結果顯示，ST-Yo-Hn-hs組耳朵纖維母細胞之促凋亡因子 (pro-apoptotic factors) 包括Caspases-3、-8和-9的蛋白相對表現量顯著（p < 0.05）低於H-hs組；反之，ST-Yo-Hn-hs組耳朵纖維母細胞之抑凋亡因子 (anti-apoptotic factors) 包括GNA14蛋白質及CRELD2和PRKCQ基因的相對表現量顯著（p < 0.05）高於H-hs組。而氧化磷酸化 (oxidative phosphorylation) 相關因子的分析結果顯示，ST-Yo-Hn-hs組耳朵纖維母細胞之GPX1基因和Complex-I、Complex-IV、CAT與PGLS蛋白的相對表現量顯著（p < 0.05）高於H-hs組。綜合之資訊說明，ICSI在ST操作前進行可以改善ST牛胚之發育能力；且使用IVF法進行ST卵母細胞受精作用較之於使用ICSI法者更顯著提高ST仔牛之懷孕率與出生率，惟此等ST仔牛之細胞質中仍存在來自供核卵母細胞的mtDNA異質性現象。此外，ST-Yo-Hn牛耳朵纖維母細胞因具有較低之促凋亡因子表現量，以及較高之氧化磷酸化與抗氧化因子的表現量，因此較之於荷蘭牛者擁有更佳之耐熱性，且此等耐熱能力至少部份源自於耐熱種台灣黃牛之細胞質。因此，利用ST技術生產含有台灣黃牛細胞質的ST荷蘭牛，可能是減輕熱緊迫對熱帶和亞熱帶地區乳牛負面影響的可行策略。
並列摘要
Holsteins are globally recognized as the predominant milk-producing dairy breed due to their high milk yields. However, the increasing global temperatures are presenting unprecedented challenges for dairy cows to adapt to elevated heat, resulting in reproductive endocrine imbalances, compromised metabolic activity, and decreased milk production and quality. With the projected increase in average annual temperatures, dairy cows are expected to experience elevated heat stress more frequently, potentially posing detrimental impacts on the dairy industry. Progress has been made in the production of heat-tolerant cows, which were produced from bovine cells originating from somatic cell nuclear transfer (SCNT) embryos utilizing the cytoplasm of Taiwan yellow cattle (Y). Nevertheless, further investigation is required to enhance oocyte developmental competence and embryonic development through spindle transfer (ST) in cattle. Therefore, two independent experiments were conducted. The initial experiment aimed to assess the viability of ST intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF) in producing embryos and calves, while the subsequent experiment aimed to elucidate the genetic and molecular factors associated with cell sensitivities in ST-Yo-Hn and Holstein (H) cattle. In the first experiment, the development of ST bovine embryos was evaluated using ICSI timing and fertilization techniques. The results revealed that ICSI-ST yielded a significantly higher blastocyst rate (24.7%) in ST embryos when compared to ST-ICSI (5.9%). Interestingly, the rate of fertilization was higher for ST-IVF (97.3%), with a polyspermy rate of 24.7%, but lower for blastocyst rate (22.7%) compared to other groups, including zona-cut oocytes (ZC), denuded oocytes (DO), and cumulus-oocyte complexes (COCs)-IVF. No significant difference was observed for in vitro embryonic development between ICSI-ST (24.5%) and ST-IVF (25.2%). Despite this, both pregnancy (46.7%) and birth rates (33.3%) were significantly higher using ST-IVF than ICSI-ST, which showed rates of 6.3% and 0.0% respectively. The concluding findings of the first study highlighted that the mitochondrial DNA (mtDNA) heteroplasmy retained from 5 claves was within a range of 2-18%. In the subsequent trial, the differentially expressed genes and genetic profiles of ST-Yo-Hn and H cattle ear fibroblasts were subjected to a comparative analysis under both heat stress (hs) conditions (42 °C, 12h) and normal conditions. The relative expression levels of pro-apoptotic factors, such as Caspases-3, -8, and -9, exhibited a statistically significant increase (P < 0.05) in the ear fibroblasts from the H-hs group in comparison to those observed in the ST-Yo-Hn-hs group. Furthermore, the ear fibroblasts from the ST-Yo-Hn-hs group showed significantly higher (P < 0.05) relative expression levels of anti-apoptotic factors, such as GNA14 protein, as well as CRELD2 and PRKCQ genes, in comparison to the H-hs group. The relative expression of oxidative phosphorylation-related factors was significantly higher (P <0.05) for GPX1 gene, and proteins of Complex-I, Complex-IV, CAT, and PGLS in the ear fibroblasts in ST-Yo-Hn-hs group than the H-hs group. The findings suggest that prioritizing the implementation of ICSI before ST could significantly enhance the embryonic development of ST bovine embryos. Moreover, the utilization of the IVF technique for ST oocyte fertilization, as opposed to ICSI, demonstrated an increase in the pregnancy and birth rates of ST calves with mtDNA heteroplasmy from donor karyoplasts. The utilization of cytoplasm from more heat-tolerant Y cattle resulted in decreased expression of pro-apoptotic factors and increased expression of oxidative phosphorylation and antioxidant factors in ST-Yo-Hn cattle, indicating enhanced thermotolerance. Therefore, implementing ST technology to produce H cattle with Y cytoplasm presents a viable strategy to alleviate the adverse effects of heat stress on dairy cattle in tropical-subtropical regions.