Eckert J, Niemann H
Institut für Tierzucht und Tierverhalten (FAL), Mariensee, 31535 Neustadt, Germany.
Theriogenology. 1996 Jul 15;46(2):307-20. doi: 10.1016/0093-691x(96)00187-2.
The purpose of our experiments was to explore the effects of platelet-derived growth factor (PDGF)-supplementation at the various steps of in vitro production of bovine embryos using protein-free media. Cumulus-oocyte-complexes (COC) were collected by slicing abattoir ovaries and then dividing the COC into 2 morphological categories. After maturation for 24 h in TCM-199 supplemented with hormones and either 20% estrous cow serum (ECS) or 1 mg/ml polyvinyl-alcohol (PVA), oocytes were co-incubated for 19 h with frozen/thawed spermatozoa from bull of proven fertility. The semen was diluted in Fert-Talp supplemented with heparin, hypotaurine and epinephrine and either 6 mg/ml bovine serum albumin (BSA) or 1 mg/ml PVA. Presumptive zygotes were transferred into embryo culture medium containing either 20% ECS or 1 mg/ml PVA for a total of 10 d. The PDGF was added at concentrations of 1, 10 or 100 ng/ml to the maturation medium (Experiment 1), fertilization medium (Experiment 2) or culture medium from Day 1 on (Experiment 3), respectively, or at 1 ng/ml PDGF to both the fertilization and culture medium from Day 3 on (Experiment 4), with each medium supplemented with PVA. Oocytes/embryos incubated in the absence of PDGF in media supplemented with either ECS or PVA served as controls. An average of 20 COC was incubated in 1 droplet under silicone oil, and each experiment contained 4 to 6 replicates. No significant differences were found among the various concentrations of PDGF, nor did PDGF-supplementation during maturation (Experiment 1) or embryo culture on Day 1 (Experiment 3) significantly affect development of oocytes/embryos (34.7 +/- 3.5 to 40.4 +/- 2.5% morulae, 11.9 +/- 2.4 to 18.8 +/- 2.5% blastocysts; and 23.2 +/- 2.3 to 27.5 +/- 3.4% morulae, 11.5 +/- 2.6 to 12.7 +/- 2.3% blastocysts, respectively; x +/- SEM). In the presence of 10 ng/ml PDGF in the fertilization medium development to morulae and blastocysts was similar to that of the ECS-group, and was higher (P < 0.05) than that of the PVA-control (ECS: 32.1 +/- 4.6 and 13.8 +/- 2.7%; PVA: 17.5 +/- 0.8 and 6.1 +/- 1.3%; PDGF: 30.6 +/- 3.0 and 14.0 +/- 2.2%, respectively). Development to morulae/blastocysts was increased, and was at the same level as in the ECS-group when the fertilization and/or embryo culture medium on Day 3 contained PDGF compared with the PVA-control group (morulae: ECS 25.3 +/- 4.4%, PVA 13.9 +/- 2.2% [P < 0.05], PDGF 16.7 +/- 3.2 to 19.1 +/- 1.1%; blastocysts: ECS 5.3 +/- 2.1%, PVA 5.0 +/- 1.7%, PDGF 7.1 +/- 1.6 to 9.1 +/- 1.7%, respectively). These results indicate that under our laboratory conditions PDGF can elevate low rates of development and the addition of PDGF to the fertilization medium enhances bovine preimplantation embryonic development. Thus, PDGF can be potentially an important factor in a completely defined medium to substitute the effects of serum.
我们实验的目的是探讨在使用无蛋白培养基体外生产牛胚胎的各个步骤中添加血小板衍生生长因子（PDGF）的效果。通过切割屠宰场卵巢收集卵丘-卵母细胞复合体（COC），然后将COC分为2种形态类别。在添加了激素以及20%发情母牛血清（ECS）或1mg/ml聚乙烯醇（PVA）的TCM-199中成熟24小时后，将卵母细胞与来自生育力已证实公牛的冻融精子共同孵育19小时。精液在添加了肝素、亚牛磺酸和肾上腺素以及6mg/ml牛血清白蛋白（BSA）或1mg/ml PVA的Fert-Talp中稀释。将推定的受精卵转移到含有20% ECS或1mg/ml PVA的胚胎培养基中培养总共10天。分别在成熟培养基（实验1）、受精培养基（实验2）或从第1天开始的培养基（实验3）中以1、10或100ng/ml的浓度添加PDGF，或者从第3天开始在受精培养基和培养基中都添加1ng/ml PDGF（实验4），每种培养基都添加PVA。在添加了ECS或PVA且未添加PDGF的培养基中孵育的卵母细胞/胚胎作为对照。平均20个COC在硅油下的1个液滴中孵育，每个实验包含4至6个重复。在不同浓度的PDGF之间未发现显著差异，在成熟期间（实验1）或第1天的胚胎培养（实验3）中添加PDGF也未显著影响卵母细胞/胚胎的发育（桑椹胚率为34.7±3.5%至40.4±2.5%，囊胚率为11.9±2.4%至18.8±2.5%；以及桑椹胚率为23.2±2.3%至27.5±3.4%，囊胚率为11.5±2.6%至12.7±2.3%，分别为x±SEM）。在受精培养基中存在10ng/ml PDGF时，发育到桑椹胚和囊胚的情况与ECS组相似，且高于PVA对照组（ECS：32.1±4.6%和13.8±2.7%；PVA：17.5±0.8%和6.1±1.3%；PDGF：30.6±3.0%和14.0±2.2%，分别）。与PVA对照组相比，当第3天的受精和/或胚胎培养基中含有PDGF时，发育到桑椹胚/囊胚的比例增加，且与ECS组处于相同水平（桑椹胚：ECS 25.3±4.4%，PVA 13.9±2.2%[P<0.05]，PDGF 16.7±3.2%至19.1±1.1%；囊胚：ECS 5.3±2.1%，PVA 5.0±1.7%，PDGF 7.1±1.6%至9.1±1.7%，分别）。这些结果表明，在我们的实验室条件下，PDGF可以提高低发育率，并且在受精培养基中添加PDGF可增强牛植入前胚胎的发育。因此，PDGF可能是完全限定培养基中替代血清作用的一个重要因素。
