體外培養之不同階段所添加抗氧化劑種類對孤雌激活及ICSI豬胚發育能力之影響

Effect of culture stages supplemented with different antioxidants on the development of parthenogenetic and ICSI porcine embryos

陳則佑(Chen Tse-Yu)

指導教授 ： 沈朋志 ； 彭劭于

國立屏東科技大學/農學院/動物科學與畜產系所/碩士(2018年)

https://doi.org/10.6346/THE.NPUST.AS.004.2018.D06

摘要

哺乳動物胚之體外生產（in vitro production, IVP）包括體外成熟（In vitro maturation, IVM）、體外受精（in vitro fertilization, IVF）及體外培養（In vitro culture, IVC）。目前於此胚體外生產期間所使用之環境氧濃度約為20%，此相較於體內約5－8%的氧濃度而言已屬高氧環境，而降低胚之後續發育能力。此外，在細胞質單一精子注射（intracytoplasmic sperm injection, ICSI）操作過程，若精子注射位置過於接近卵母細胞核，將造成ICSI胚後續發育能力之降低。惟抗氧化劑種類及培養階段對豬卵母細胞及胚後續發育能力，以及精子注射位置對ICSI豬胚發育能力之影響，均尚未進行詳細之探討。基於此，本研究中將以豬卵母細胞作為試驗材料，首先探討此等卵母細胞於體外成熟及體外培養階段添加不同濃度之glutathione（GSH）及β-mercaptoethanol（β-ME）（研究一）對豬卵母細胞孤雌激活後發育能力之影響；於研究二，則以研究一所得較適當濃度之GSH或β-ME進行豬卵母細胞及ICSI豬胚之體外培養條件，進一步評估培養液種類及精子注射位置對ICSI豬胚體外發育能力之影響。於研究一IVM階段之結果顯示，IVM培養液中添加0－5 mM GSH之豬卵母細胞於激活後均具有相近之囊胚率（15.8－25.0%）；而IVM培養液中添加10 mM -ME之豬卵母細胞於激活後之囊胚率（35.9%）則顯著高於（P < 0.05）IVM培養液中未添加-ME之對照組（20.5%），但IVM培養液中添加25 mM（23.4%）及50 mM（23.5%）-ME之囊胚率與對照組間均無顯著（P > 0.05）差異；惟IVM培養液中添加1 mM GSH及10 mM -ME之豬卵母細胞於激活後則有相近之囊胚率（18.7 vs. 24.4%）。在IVC階段之結果則顯示，於IVM階段卵母細胞皆培養於10 mM -ME條件下，IVC培養液中添加0.5 mM GSH組孤雌激活豬胚之囊胚率（41.7%）雖與未添加GSH之對照組（32.4%）無顯著（P > 0.05）差異，但均顯著高於（P < 0.05）IVC培養液中添加1 mM（27.1%）、5 mM（26.6%）GSH處理組之囊胚率；惟於IVC培養液中添加10－50 mM -ME（12.1－20.3%）與對照組（22.4%）間孤雌激活豬胚之囊胚率均無顯著（P > 0.05）差異，且IVC培養液中添加0.5 mM GSH及10 mM -ME之孤雌激活豬胚亦均有相近之囊胚率（15.6 vs. 21.1%）。研究二之結果顯示，卵母細胞經含10 mM -ME 之NCSU-23、NCSU-23-hCG及TCM-199-hCG培養液IVM後，孤雌激活胚之囊胚發育率（14.1－19.4%）於上述3處理組別間均無顯著（P > 0.05）差異，但以TCM-199-hCG培養液組具最高之囊胚率（19.4%）。在比較精子注射位置及精液靜置後上下層精子作為注射來源精子對ICSI胚發育能力之影響結果顯示，將精子注射於靠近卵母細胞第一極體位置所產製之ICSI豬胚於激活處理後之囊胚率顯著低於（P < 0.05）精子注射位置遠離卵母細胞第一極體者（3.1 vs. 15.0%）。惟在遠離卵母細胞第一極體注射精子及選用具活動力之精子條件下，不論選取未於上層（13.8%）或下層（12.4%）之精子，所產製ICSI豬胚經激活後均具有相近之囊胚率（P > 0.05）。綜合上述之結果說明，豬卵母細胞之IVM階段及胚培養之IVC階段均可於培養液中添加10 mM -ME，藉以提升孤雌激活豬胚之體外囊胚率；而在產製ICSI豬胚時，精子注射位置宜遠離卵母細胞之第一極體，但若選取具有活動力精子時，則靜置後處於上下層之精子皆可使用。

關鍵字

穀胱甘肽 ； 細胞質單一精子注 ； 體外培養階段 ； 2-巰基乙醇 ； 卵母細胞 ； 孤雌激活胚 ； 豬

並列摘要

In vitro production (IVP) of mammalian embryos are inclusive of in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC).At present, the ambient oxygen concentration used during the in vitro production of this embryos are about 20%, which is already a high oxygen environment comparing with the oxygen concentration of about 5 to 8% in vivo, and reduce the development capability of the embryo. In addition, during intracytoplasmic sperm injection (ICSI), if the sperm injection is too close to the oocytes nucleus, the development of ICSI embryos will be reduced. However, the effects of antioxidant types and culture stages to the subsequent development of oocytes and embryos in porcine, as well as sperm injection location on the development of embryos in ICSI porcine have not yet been discussed in detail. Based on this aspect, porcine oocytes were used as experimental materials in this study. Firstly, different concentration of glutathione (GSH) and β-mercaptoethanol (β-ME) are studied in in vitro maturation and in vitro culture on the developmental ability of porcine oocytes after parthenogenetic activation. In study 2, the in vitro culture conditions of porcine oocytes and ICSI porcine embryos were explored with the specific concentration of GSH or β-ME obtained from study 1 to further evaluate the effects of both types and sperm injection position for the in vitro development ability of ICSI porcine embryos. The results of IVM stage showed that IVM medium with 0-5 mM GSH group porcine oocytes shared similar blastocyst rates (15.8-25.0%) after parthenogenetic activation. IVM medium with 10 μM β-ME, the blastocyst rate (35.9%) was significantly higher (P < 0.05) than control group (20.5%) and which was not supplemented with β-ME. However, the blastocyst rates of IVM medium with 25 μM (23.4%), 50 μM β-ME and control group (23.5%) had no significant differences (P > 0.05). IVM medium with 1 mM GSH and 10 μM β-ME of porcine oocytes which are activated sharing similar blastocyst rates (24.4% vs. 18.7). The results at the IVC stage showed that in the IVM stage oocytes were cultured at 10 μM β-ME condition, the blastocyst rate of IVC culture medium with 0.5 mM GSH (41.7%) after parthenogenetic activated porcine embryos showing no significant difference (P > 0.05) from control group (32.4%), but significantly (P < 0.05) higher than IVC medium with 1 mM (27.1%) and 5 mM (26.6%) GSH treatment group. However, the blastocyst rate of IVC medium with 10 to 50 μM β-ME (12.1-20.3%) and control group (22.4%) after parthenogenetic activation of porcine embryo shared no significant difference (P > 0.05), and IVC medium with 0.5 mM GSH and 10 μM β-ME after parthenogenetic activation of porcine embryo also possess similar blastocyst rate (21.1% vs. 15.6). The results of Study 2 show that oocytes culture in the IVM medium with 10 μM β-ME NCSU-23, NCSU-23-hCG, and TCM-199-hCG medium, The blastocyst development rate of parthenogenetic embryos (14.1-19.4%) had no significantly different (P > 0.05) among the above 3 treatment groups, but in TCM-199-hCG group shared the highest blastocyst rate (19.4%). After comparison of sperm injection position and semen let stand upper and lower sperm as a source of ICSI sperm injection on embryo development capability. According to the results, the blastocyst rate of ICSI porcine embryos produced by injecting sperm near the first polar body of oocytes after activation was significantly lower than(P < 0.05) that sperm injection position away from the first polar body of oocytes (3.1 vs. 15.0%). Whereas, under the condition of sperm injection position away from the first polar body of oocytes and selection of the sperm activity, no matter what sort of choice is located in the upper (13.8%) or lower (12.4%) of the sperm, porcine production by ICSI embryos have similar (P > 0.05) blastocyst rate after activation. Taken together, both the IVM stage of porcine oocytes and the IVC stage of embryo culture may be supplemented with 10 μM β-ME to the culture medium to increase the in vitro blastocyst rate of parthenogenetic activation porcine embryos; However, in the production of ICSI porcine embryos, the sperm injection position should be away from the first polar body of the oocyte, but if active sperm is selected, the sperm in the upper and lower layers after standing can be implemented.

並列關鍵字

Glutathione ； ICSI ； In vitro culture stage ； β-mercaptoethanol ； oocytes ； parthenogenetic embryos ； porcine

參考文獻

沈朋志、鄭三寶、劉炳燦、許勝富、陳建宇、顏淑卿。1998。取卵時間與小鼠及家兔卵母細胞第一極體形態之完整性和染色體之相對位置之關係。中畜會誌。27：251－262。

沈朋志。2003。經由成年體細胞核轉置技術產製轉基因複製家畜之可行性。國立臺灣大學畜產學研究所博士論文。

Abeydeera, L. R., W. H. Wang, T. C. Cantley, R. S. Prather, and B. N. Day. 1998. Presence of β–mercaptoethanol can increase the glutathione content of pig oocytes matured in vitro and the rate of blastocyst development after in vitro fertilization. Theriogenoloy. 50: 747-756.

Abou-haila, A., and D. R. Tulsiani. 2009. Signal transduction pathways that regulate sperm capacitation and the acrosome reaction. Arch. Biochem. Biophys. 485: 72-81.

Agarwal, A., S. Gupta, and R. Sharma. 2005. Oxidative stress and its implications in female infertility - a clinician's perspective. Reprod. Biomed. Online. 11: 641-650.