Cadenas J, Leiva-Revilla J, Vieira L A, Apolloni L B, Aguiar F L N, Alves B G, Lobo C H, Rodrigues A P R, Apgar G A, Smitz J, Figueiredo J R, Maside C
Laboratory of Manipulation of Oocytes and Preantral Follicles (LAMOFOPA), State University of Ceará, Fortaleza, CE, Brazil.
Laboratory of Animal Physiology, Department of Animal Science, Federal University of Ceará, Fortaleza, CE, Brazil.
Theriogenology. 2017 Jan 1;87:321-332. doi: 10.1016/j.theriogenology.2016.09.008. Epub 2016 Sep 13.
The aim of the present study was to evaluate the effect of growth hormone (GH) and vascular endothelial growth factor (VEGF) added alone, sequentially or in combination, in the presence of insulin at physiological concentration (10 ng/mL) on the IVC of two different follicular categories: preantral (experiment 1; Exp.1) and early antral (experiment 2; Exp.2). Isolated follicles were individually cultured for 24 (Exp.1) and 18 days (Exp.2) in the following treatments: αMEM (Control), or Control medium supplemented with 50 ng/mL GH (GH), 100 ng/mL VEGF (VEGF), the combination of both (GH + VEGF), GH during the first 12 days and VEGF from Day 12 until the end of the culture (GH/VEGF) and vice versa (VEGF/GH). At the end of the culture, cumulus-oocyte complexes from in vitro-grown follicles were recovered and subjected to IVM. The following end points were evaluated: Follicle morphology, growth rates and antrum formation, production of estradiol, progesterone and testosterone, oocyte viability and meiotic stage, as well as relative expression of LHR, Amh, HAS2, PTGS2, CYP17, CYP19A1, and 3βHSD. A considerable amount of viable fully grown oocytes were recovered after the IVC of early antral follicles in all treatments. Nevertheless, the GH treatment presented the highest percentage of fully grown oocytes (60%), mean oocyte diameter (117.74 ± 2.61 μm), and meiotic resumption (50%). Furthermore, GH treatment produced higher (P < 0.05) rates of metaphase II oocytes than all the other treatments, and similar LHR, Amh, and PTGS2 transcript levels to in vivo. Contrary to early antral follicles, preantral follicles were not affected by medium supplementation. In conclusion, the addition of GH to a culture medium containing physiological concentrations of insulin improves oocyte growth and maturation after the IVC of goat early antral follicles.
本研究的目的是评估在生理浓度(10 ng/mL)胰岛素存在的情况下,单独添加、依次添加或联合添加生长激素(GH)和血管内皮生长因子(VEGF),对两种不同卵泡类型:腔前卵泡(实验1;Exp.1)和早期有腔卵泡(实验2;Exp.2)体外培养(IVC)的影响。将分离的卵泡分别在以下处理中培养24天(Exp.1)和18天(Exp.2):αMEM(对照),或添加50 ng/mL GH的对照培养基(GH)、100 ng/mL VEGF(VEGF)、两者组合(GH + VEGF)、前12天添加GH,从第12天到培养结束添加VEGF(GH/VEGF),反之亦然(VEGF/GH)。培养结束时,回收体外培养卵泡的卵丘-卵母细胞复合体并进行体外成熟(IVM)。评估以下终点:卵泡形态、生长速率和腔形成、雌二醇、孕酮和睾酮的产生、卵母细胞活力和减数分裂阶段,以及促黄体生成素(LHR)、抗缪勒氏管激素(Amh)、透明质酸合酶2(HAS2)、前列腺素内过氧化物合酶2(PTGS2)、细胞色素P450 17α-羟化酶(CYP17)、细胞色素P450 19A1(CYP19A1)和3β-羟基类固醇脱氢酶(3βHSD)的相对表达。在所有处理中,早期有腔卵泡体外培养后回收了大量存活的完全成熟卵母细胞。然而,GH处理组的完全成熟卵母细胞百分比最高(60%),平均卵母细胞直径最大(117.74 ± 2.61 μm),减数分裂恢复率最高(50%)。此外,GH处理组产生的中期II期卵母细胞率高于所有其他处理组,且LHR、Amh和PTGS2转录水平与体内相似(P < 0.05)。与早期有腔卵泡相反,腔前卵泡不受培养基添加物的影响。总之,在含有生理浓度胰岛素的培养基中添加GH可改善山羊早期有腔卵泡体外培养后的卵母细胞生长和成熟。