Sá N A R, Araújo V R, Correia H H V, Ferreira A C A, Guerreiro D D, Sampaio A M, Escobar E, Santos F W, Moura A A, Lôbo C H, Ceccatto V M, Campello C C, Rodrigues A P R, Leal-Cardoso J H, Figueiredo J R
Laboratory of Manipulation of Oocytes and Preantral Follicles, Faculty of Veterinary Medicine, State University of Ceará, Fortaleza, CE, Brazil.
Laboratory of Manipulation of Oocytes and Preantral Follicles, Faculty of Veterinary Medicine, State University of Ceará, Fortaleza, CE, Brazil.
Theriogenology. 2017 Feb;89:226-234. doi: 10.1016/j.theriogenology.2015.12.014. Epub 2015 Dec 28.
The aim of this study was to investigate the effect of three concentrations of anethole (30, 300, and 2000 μg/mL) on survival, antrum formation, follicular diameter, and oocyte maturation in the caprine species. The study also evaluated the effects of anethole on transcripts of ICAM-1, CAV-1, TIMP-2, and PAI-1 genes and levels of reactive oxygen species (ROS) in isolated goat preantral ovarian follicles before and after in vitro culture for 18 days. Preantral follicles were isolated from goat ovaries and individually cultured in alpha minimum essential medium modified (α-MEM), defined as the control treatment, α-MEM supplemented with ascorbic acid at a concentration of 100 μg/mL (AA), or α-MEM supplemented with three different concentrations of anethole (30, 300, 2000 μg/mL) for a period of 18 days. Treatments were named as α-MEM, AA, AN30, AN300, and AN2000, respectively. After culture, the follicles were opened, the cumulus oocytes complex (COCs) were removed and matured in vitro. The walls of the follicles were used for the quantitation of mRNA by quantitative real-time polymerase chain reaction. Finally, the medium collected at the end of culture was used for the measurements of ROS. After 18 days of culture, the AA treatment showed the percentage of intact follicles and follicular diameter significantly higher compared with the other treatments. However, daily growth rate, antrum formation, and also oocyte diameter were similar among the treatments. In addition, compared with AA, the rate of oocytes for in vitro maturation (diameter ≥ 110 μm) and the meiosis resumption rate were significantly higher in the treatments AN30 and AN2000, respectively. When assessing gene related to remodeling of the basement membrane, significant differences in mRNA levels for ICAM-1, CAV-1, TIMP-2, and PAI-1 were observed in comparison with Day 0, i.e., in the noncultured control. In addition, the ROS from Day 12, all treatments with the addition of anethole have significantly lower values of ROS than α-MEM and AA. In conclusion, the addition of anethole to the in vitro culture medium was able to improve the development of goat preantral follicles by reducing concentrations of ROS and increasing the percentage of oocytes able to resume meiosis.
本研究的目的是调查三种浓度的茴香脑（30、300和2000μg/mL）对山羊物种的存活、窦腔形成、卵泡直径和卵母细胞成熟的影响。该研究还评估了茴香脑对体外培养18天前后分离的山羊腔前卵泡中ICAM-1、CAV-1、TIMP-2和PAI-1基因转录本以及活性氧（ROS）水平的影响。从山羊卵巢中分离出腔前卵泡，并分别在改良的α-最低必需培养基（α-MEM）中培养，定义为对照处理；在α-MEM中添加浓度为100μg/mL的抗坏血酸（AA）；或在α-MEM中添加三种不同浓度的茴香脑（30、300、2000μg/mL），培养18天。处理分别命名为α-MEM、AA、AN30、AN300和AN2000。培养后，打开卵泡，取出卵丘卵母细胞复合体（COC）并进行体外成熟培养。卵泡壁用于通过定量实时聚合酶链反应定量mRNA。最后，将培养结束时收集的培养基用于测量ROS。培养18天后，AA处理组的完整卵泡百分比和卵泡直径显著高于其他处理组。然而，各处理组的每日生长速率、窦腔形成以及卵母细胞直径相似。此外，与AA相比，AN30和AN2000处理组的体外成熟卵母细胞（直径≥110μm）率和减数分裂恢复率分别显著更高。在评估与基底膜重塑相关的基因时，与第0天（即未培养的对照）相比，观察到ICAM-1、CAV-1、TIMP-2和PAI-1的mRNA水平存在显著差异。此外，从第12天起，所有添加茴香脑的处理组的ROS值均显著低于α-MEM和AA。总之，在体外培养基中添加茴香脑能够通过降低ROS浓度和增加能够恢复减数分裂的卵母细胞百分比来改善山羊腔前卵泡的发育。
