Pavlok A, Kubelka M, Peknicová J
Institute of Animal Physiology and Genetics, Libechov, Czech Republic.
Zygote. 2001 Feb;9(1):25-38. doi: 10.1017/s0967199401001046.
In this paper the effects of capacitation and fertilisation stimulating compounds (heparin, caffeine, glucose, D-penicillamine, bovine serum (BOS), bovine serum albumin (BSA), polyvinyl alcohol (PVA)) were analysed in several in vitro fertilisation protocols. Attention was paid to the rate of penetrated oocytes, kinetics of penetration and to polyspermic fertilisation. Cryopreserved bovine sperm and in vitro matured bovine oocytes were used throughout all the fertilisation experiments. As detected in the first 8 h fertilisation experiment with non-incubated sperm, the supplementation of medium with heparin, BOS and glucose supported the fertilisation rate most effectively (100%), including the kinetics of pronuclei formation (52.4%). The absence of BOS resulted in a decreased fertilisation rate (62.7%) as well as a delay in pronuclei formation (13.6%), similar to that after substitution of heparin with caffeine (73.0% and 25.4%, respectively). The penetration rate in the control medium with BOS (without heparin and caffeine) was surprisingly high, especially in medium without glucose (62.2%). The positive effect of glucose on sperm penetration was observed mainly in a chemically defined medium with PVA. High polyspermy rates were observed throughout all experiments in the media containing heparin or caffeine and BOS as the macromolecular component. D-Penicillamine was not shown to be a fertilisation-stimulating molecule. However, as detected in the second experiment in which oocytes were fertilised with 5 h incubated sperm, its positive effect on the prolongation of a fertile life span of cryopreserved spermatozoa was significant. The presence of either caffeine or heparin in the fertilisation medium (FM) with BOS during sperm incubation induced tyrosine phosphorylation of an approximately 90 kDa protein, detected after 5 h of sperm incubation. The absence of BOS reduced tyrosine phosphorylation of this protein in fertilisation medium with heparin. The percentage of motile spermatozoa and those with intact acrosomes were monitored throughout all experiments.
在本文中，我们在多种体外受精方案中分析了获能和受精刺激化合物（肝素、咖啡因、葡萄糖、D-青霉胺、牛血清（BOS）、牛血清白蛋白（BSA）、聚乙烯醇（PVA））的作用。重点关注了卵母细胞的穿透率、穿透动力学以及多精受精情况。在所有受精实验中均使用了冷冻保存的牛精子和体外成熟的牛卵母细胞。正如在第一个对未孵育精子进行的8小时受精实验中所检测到的，向培养基中添加肝素、BOS和葡萄糖对受精率的支持最为有效（100%），包括原核形成的动力学（52.4%）。缺乏BOS会导致受精率降低（62.7%）以及原核形成延迟（13.6%），这与用咖啡因替代肝素后的情况类似（分别为73.0%和25.4%）。在含有BOS的对照培养基（不含肝素和咖啡因）中的穿透率出奇地高，尤其是在不含葡萄糖的培养基中（62.2%）。葡萄糖对精子穿透的积极作用主要在含有PVA的化学成分明确的培养基中观察到。在所有实验中，在含有肝素或咖啡因以及作为大分子成分的BOS的培养基中均观察到了较高的多精受精率。D-青霉胺未被证明是一种受精刺激分子。然而，正如在第二个实验中所检测到的，用孵育5小时的精子使卵母细胞受精时，它对延长冷冻保存精子的可育寿命具有显著的积极作用。在精子孵育期间，在含有BOS的受精培养基（FM）中存在咖啡因或肝素会诱导一种约90 kDa蛋白质的酪氨酸磷酸化，这在精子孵育5小时后可以检测到。在含有肝素的受精培养基中，缺乏BOS会降低这种蛋白质的酪氨酸磷酸化。在所有实验中均监测了活动精子和顶体完整精子的百分比。
