Maciel V L, Caldas-Bussiere M C, Silveira V, Reis R S, Rios A F L, Paes de Carvalho C S
Laboratório de Reprodução e Melhoramento Genético Animal, Centro de Ciências e Tecnologias Agropecuárias (CCTA), Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes, RJ, Brazil.
Laboratório de Biotecnologia, Centro de Biociências e Biotecnologia (CBB), Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes, RJ, Brazil; Unidade de Biologia Integrativa, Setor de Genômica e Proteômica, Laboratório de Biotecnologia, Centro de Biociências e Biotecnologia (CBB), Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes, RJ, Brazil.
Theriogenology. 2018 Oct 1;119:1-9. doi: 10.1016/j.theriogenology.2018.06.018. Epub 2018 Jun 27.
The aim of this work was to evaluate the proteomic changes that occurred in the frozen-thawed bovine spermatozoa after the addition of l-arginine (L-arg) during in vitro sperm capacitation. Aspects related to the sperm capacitation pattern like membrane integrity, mitochondrial activity, sperm motility and vigor, and the sperm proteome were determined. These were respectively assessed by chlortetracycline staining, H342/PI, JC-1, light microscopy, and the proteomic abundance by nUPLC-MS/MS analysis. Frozen-thawed sperm from three Nellore bulls were capacitated in vitro for 3 h in sp-TALP medium supplemented with 20 μg/mL heparin (Control) or with 20 μg/mL heparin plus 1 mM L-arg (L-arg group). Data were subjected to analysis of variance and means compared by SNK test at 5% probability. When compared to Control, the percentage of sperm motility was higher in the L-arg group (P < 0.05). For test data after 3 h of incubation, sperm capacitated with L-arg showed higher membrane integrity and mitochondrial potential when compared to Control (P < 0.05). Moreover, we observed an increase in the percentage of capacitated sperm pattern (P < 0.05). Protein abundance analysis identified 367 proteins. Forty proteins were differentially abundant between Control and L-arg group (P < 0.05), of which 11 were up-regulated, and 29 were down-regulated in L-arg group. In addition, we observed that one protein was uniquely abundant in the L-arg group. Our findings indicate that the addition of L-arg to the culture medium presented a differential protein abundance pattern and increased the bovine frozen-thawed sperm quality and the percentage of capacitated sperm. The proteomic changes observed may be linked to the molecular mechanisms involved in the action of L-arg on the in vitro sperm capacitation of cattle.
本研究的目的是评估在体外精子获能过程中添加L-精氨酸（L-arg）后，冻融牛精子发生的蛋白质组学变化。测定了与精子获能模式相关的方面，如膜完整性、线粒体活性、精子活力和活力，以及精子蛋白质组。这些分别通过金霉素染色、H342/PI、JC-1、光学显微镜进行评估，并通过nUPLC-MS/MS分析评估蛋白质组丰度。来自三头内洛尔公牛的冻融精子在补充有20μg/mL肝素的sp-TALP培养基（对照）或补充有20μg/mL肝素加1mM L-arg的sp-TALP培养基（L-arg组）中体外获能3小时。数据进行方差分析，并通过SNK检验以5%的概率比较均值。与对照相比，L-arg组的精子活力百分比更高（P<0.05）。对于孵育3小时后的测试数据，与对照相比，用L-arg获能的精子显示出更高的膜完整性和线粒体电位（P<0.05）。此外，我们观察到获能精子模式的百分比增加（P<0.05）。蛋白质丰度分析鉴定出367种蛋白质。对照和L-arg组之间有40种蛋白质差异丰富（P<0.05），其中11种在L-arg组中上调，29种在L-arg组中下调。此外，我们观察到一种蛋白质在L-arg组中独特丰富。我们的研究结果表明，在培养基中添加L-arg呈现出差异蛋白质丰度模式，并提高了牛冻融精子的质量和获能精子的百分比。观察到的蛋白质组学变化可能与L-arg对牛体外精子获能作用所涉及的分子机制有关。
