Maturation in vitro of pig oocytes in protein-free culture media: Fertilization and subsequent embryo development in vitro
Maturation in vitro of pig oocytes in protein-free culture media: Fertilization and subsequent embryo development in vitro
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DOI：
10.1095/biolreprod58.5.1316
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发表时间：
1998-05-01
影响因子：
3.6
通讯作者：
Day, BN
中科院分区：
文献类型：
--
作者：
Abeydeera, LR;Wang, WH;Day, BN
本研究试图建立猪卵母细胞的无蛋白体外成熟(PF)系统，并对体外受精后的胚胎发育进行检测。在实验1中，实验分为4种：1)对照组：北卡罗来纳州立大学(NCSU)23+10%猪卵泡液；2)PF-NCSU：NCSU 23+0.1%聚乙烯醇(PVA)+1%氨基酸；3)PF-Medice：组织培养基199+PVA；4)PF-WM：PF-Wayuss MB 752/1培养液+PVA。将卵母细胞分别在分别含有CG和hCG(10IU/ml)的培养液中培养20~22h，然后在不添加激素的情况下再培养20~22h，培养后测定卵丘的扩张程度和核成熟的频率。将部分卵母细胞与冷冻解冻后的精子在含有咖啡因和BSA的改良Tris缓冲液中共孵育5~6h。在实验2中，卵母细胞在对照组、PF-Tcm和PF-WM中成熟，体外受精，在NSCU23+BSA中培养144 h。在PF-NCSU中，进入中期II期的卵母细胞(45%比80-85%)要少(P<；0.01)。在对照组中成熟的卵母细胞表现出最大的卵丘扩张，其次是PF-Tcm和PF-WM，而在PF-NCSU中成熟的卵母细胞表现出极轻微的扩张。在PF-NCSU中成熟的卵母细胞的穿透率(p<；0.05)低于对照组(59%vs.81%)。与对照组(96%)和PF-Tcm组(93%)相比，PF-WM组(65%)的雄性原核形成率较低。与对照组相比，在PF-WM中成熟的卵母细胞的卵裂率也显著降低(p<；0.05)。当卵母细胞成熟时，对照组(22%)和PF-Medici组(13%)的胚胎发育到囊胚期的比例相似。这些结果表明，猪卵母细胞可以在无蛋白的培养液中成功成熟，并随后发育到囊胚期。
In the present study, attempts were made to develop a protein-free (PF) in vitro maturation (IVM) system for pig oocytes and to examine subsequent embryo development after in vitro fertilization. In experiment 1, four IVM media were tested: 1) control: North Carolina State University (NCSU) 23+10% porcine follicular fluid; 2) PF-NCSU: NCSU 23+0.1% polyvinyl alcohol (PVA)+1% amino acids; 3) PF-TCM: Tissue culture medium (TCM) 199+PVA; and 4) PF-WM: PF-Waymouth MB 752/1 medium (WM)+PVA. Oocytes were cultured in the respective media containing eCG and hCG (10 IU/ml each) for 20-22 h and then without hormonal supplements for an additional 20-22 h. After culture, the degree of cumulus expansion and frequency of nuclear maturation were determined. Some oocytes were coincubated with frozen-thawed spermatozoa for 5-6 h in modified Tris-buffered medium containing caffeine and BSA. In experiment 2, oocytes were matured in control, PF-TCM, and PF-WM, fertilized in vitro, and cultured for 144 h in NSCU 23+BSA. Fewer (p < 0.01) oocytes reached metaphase II stage in PF-NCSU (45% vs. 80-85%) than in the other media. Oocytes matured in control medium showed the most cumulus expansion, followed by those in PF-TCM and PF-WM; those in PF-NCSU showed very slight expansion. A lower (p < 0.05) penetration rate was obtained for oocytes matured in PF-NCSU than in the control medium (59% vs. 81%). In contrast to those in control (96%) and PF-TCM (93%), oocytes in PF-WM (65%) showed a lower male pronuclear formation. Compared to that in the control, a significantly lower (p < 0.05) cleavage rate was also observed for oocytes matured in PF-WM. Similar proportions of embryos developed to the blastocyst stage when oocytes were matured in control (22%) and PF-TCM (13%). These results indicate that pig oocytes can be successfully matured in a protein-free medium with subsequent development to the blastocyst stage.