Niu Yuyu, Greube Alexa, Ji Weizhi, Jewgenow Katarina
Institute for Zoo and Wildlife Research, PF 601103, D-10252 Berlin, Germany.
Theriogenology. 2006 Sep 1;66(4):989-95. doi: 10.1016/j.theriogenology.2006.02.043. Epub 2006 Apr 18.
The present study aimed to establish a sensitive in vitro assay to assess the binding capacity of cat spermatozoa. Cat oocytes and epididymal sperm cells were isolated from gonads and cultured for in vitro fertilization. Before fertilization, the sperm cells were incubated either in 10 microM green dye Fluo-3-AM or 10 microM orange dye CellTracker Orange CMTMR (Molecular Probes), respectively. After removing the dyes by washing, sperm cells stained with each dye were added to medium drops containing oocytes in various proportions and cultured for 16 h at 37 degrees C, 5% CO(2). The oocytes were examined using fluorescence microscopy. Sperm bound to oocytes, and stained with different colors, were counted. When fresh epididymal sperm were mixed in at a specific proportion, the number of sperm bound to the zona pellucida (ZP) of oocytes reflected the proportion of differently colored sperm in the medium. This indicated that neither dye influenced the binding capacity of cat sperm. Mixing fresh and cryopreserved sperm, however, resulted in a higher number of fresh sperm bound to the oocyte surface in comparison to frozen-thawed sperm. Also, the pre-incubation of cat sperm cells with ZP derived peptide reduced the sperm binding capacity by 40%. In conclusion, the presented sperm competition assay allows assessment of fertilizing capacity of cat spermatozoa in vitro when a mixture of two different populations is used. The applied supravital fluorescence dyes do not affect motility and binding capacity of sperm cells and were clearly distinguishable under fluorescence microscopy. We demonstrate that the assay can be used to study the impact of sperm treatment, such as cryopreservation or pre-incubation in bioactive peptides, on fertilizing capacity.
本研究旨在建立一种灵敏的体外检测方法，以评估猫精子的结合能力。从性腺中分离出猫卵母细胞和附睾精子细胞，并进行体外受精培养。在受精前，精子细胞分别在10微摩尔绿色染料Fluo-3-AM或10微摩尔橙色染料CellTracker Orange CMTMR（分子探针公司）中孵育。通过洗涤去除染料后，将用每种染料染色的精子细胞以不同比例添加到含有卵母细胞的培养基滴中，并在37℃、5%二氧化碳条件下培养16小时。使用荧光显微镜检查卵母细胞。对与卵母细胞结合并染有不同颜色的精子进行计数。当以特定比例混入新鲜附睾精子时，与卵母细胞透明带（ZP）结合的精子数量反映了培养基中不同颜色精子的比例。这表明两种染料均未影响猫精子的结合能力。然而，将新鲜精子与冷冻保存的精子混合时，与冻融精子相比，结合到卵母细胞表面的新鲜精子数量更多。此外，猫精子细胞与ZP衍生肽预孵育会使精子结合能力降低40%。总之，当使用两种不同群体的混合物时，所呈现的精子竞争检测方法可用于体外评估猫精子的受精能力。所应用的超活荧光染料不影响精子细胞的活力和结合能力，并且在荧光显微镜下可清晰区分。我们证明该检测方法可用于研究精子处理（如冷冻保存或在生物活性肽中预孵育）对受精能力的影响。
