Abeydeera L R, Wang W H, Cantley T C, Rieke A, Murphy C N, Prather R S, Day B N
Department of Animal Sciences, University of Missouri, Columbia, MO 65211, USA.
Theriogenology. 2000 Sep 15;54(5):787-97. doi: 10.1016/S0093-691X(00)00390-3.
This study examined the ability of epidermal growth factor (EGF) to improve the developmental competence of pig oocytes matured in a protein-free (PF) in vitro maturation (IVM) system. Oocyte maturation was done in one of three media: 1. PF-TCM: tissue culture medium (TCM) 199 + 0.1% polyvinylalcohol (PVA); 2. PF-TCM+EGF: PF-TCM + 10 ng/ml EGF; and 3. +ve CONT: North Carolina State University (NCSU) 23 medium + 10% porcine follicular fluid. All media contained 0.57 mM cysteine. Hormonal supplements, 0.5 microg/mL LH and 0.5 microg/mL FSH, were present only for the first half (20 to 22 h) of the culture period. After maturation, oocytes were co-incubated with frozen-thawed spermatozoa for 5 to 6 h and transferred to embryo culture medium, NCSU 23 containing 0.4% BSA, for 144 h. In Experiment 1, differences in cumulus expansion were observed for oocytes matured in +ve CONT (Category 4), PF-TCM (Category 2) and PF-TCM+EGF (Category 3). However, no significant differences in nuclear maturation to metaphase II stage were observed. In Experiment 2, no differences in fertilization parameters were observed. Significant (P < 0.01) differences in cleavage rates were observed among the three media for a proportion of the oocytes matured (52, 60 and 69% in PF-TCM, PF-TCM+EGF, and +ve CONT, respectively). Oocytes matured in PF-TCM showed the lowest (P < 0.01) blastocyst development (22%). However, the same rate of blastocyst development was obtained for +ve CONT (37%) and PF-TCM+EGF (37%). Blastocyst cell numbers were significantly higher when oocytes were matured in the presence of EGF (26 vs. 37 to 41). In Experiment 3, oocytes matured in PF-TCM+EGF had a significantly (P < 0.05) higher intracellular glutathione (GSH) concentration (5.9 vs. 11.4 pmol/oocyte) compared with PF-TCM. Twenty-two of 25 embryo transfer recipients became pregnant (Experiment 4). Four animals returned to estrus in within 60 days. Six pregnant animals slaughtered at 26 to 45 days had 43 fetuses (range: 4 to 12) and the remaining 12 animals farrowed 82 piglets (range: 3 to 12). These results indicate that EGF enhances the developmental competence of pig oocytes matured in a protein-free culture medium which is correlated with higher GSH level in oocytes. Birth of piglets indicate that embryos derived from oocytes matured in the presence of EGF are viable.
本研究检测了表皮生长因子（EGF）提高在无蛋白（PF）体外成熟（IVM）系统中成熟的猪卵母细胞发育能力的作用。卵母细胞在以下三种培养基之一中进行成熟培养：1. PF-TCM：组织培养液（TCM）199 + 0.1%聚乙烯醇（PVA）；2. PF-TCM+EGF：PF-TCM + 10 ng/ml EGF；3. +ve CONT：北卡罗来纳州立大学（NCSU）23培养基 + 10%猪卵泡液。所有培养基均含有0.57 mM半胱氨酸。仅在培养期的前半段（20至22小时）添加激素补充剂，即0.5 μg/mL促黄体生成素（LH）和0.5 μg/mL促卵泡素（FSH）。成熟后，将卵母细胞与冻融精子共同孵育5至6小时，然后转移至含有0.4%牛血清白蛋白（BSA）的胚胎培养液NCSU 23中培养144小时。在实验1中，观察到在+ve CONT（4级）、PF-TCM（2级）和PF-TCM+EGF（3级）中成熟的卵母细胞在卵丘扩展方面存在差异。然而，在核成熟至中期II期方面未观察到显著差异。在实验2中，未观察到受精参数的差异。对于部分成熟的卵母细胞，三种培养基之间在卵裂率上存在显著（P < 0.01）差异（PF-TCM、PF-TCM+EGF和+ve CONT中分别为52%、60%和69%）。在PF-TCM中成熟的卵母细胞囊胚发育率最低（P < 0.01）（22%）。然而，+ve CONT（37%）和PF-TCM+EGF（37%）的囊胚发育率相同。当卵母细胞在EGF存在下成熟时，囊胚细胞数量显著增加（26个对37至41个）。在实验3中，与PF-TCM相比，在PF-TCM+EGF中成熟的卵母细胞细胞内谷胱甘肽（GSH）浓度显著更高（P < 0.05）（5.9对11.4 pmol/卵母细胞）。25只胚胎移植受体中有22只怀孕（实验4）。4只动物在60天内返情。6只在26至45天屠宰的怀孕动物有43只胎儿（范围：4至12只），其余12只动物产下82头仔猪（范围：3至12头）。这些结果表明，EGF可提高在无蛋白培养基中成熟的猪卵母细胞的发育能力，这与卵母细胞中较高的GSH水平相关。仔猪的出生表明，在EGF存在下成熟的卵母细胞来源的胚胎是有活力的。
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