Ferreira A C A, Maside C, Sá N A R, Guerreiro D D, Correia H H V, Leiva-Revilla J, Lobo C H, Araújo V R, Apgar G A, Brandão F Z, Figueiredo J R, Campello C C
Laboratory of Manipulation of Oocytes and Preantral Follicles, Faculty of Veterinary, State University of Ceará, Fortaleza, CE, Brazil.
Laboratory of Manipulation of Oocytes and Preantral Follicles, Faculty of Veterinary, State University of Ceará, Fortaleza, CE, Brazil.
Anim Reprod Sci. 2016 Feb;165:1-10. doi: 10.1016/j.anireprosci.2015.10.010. Epub 2015 Nov 1.
The aim of this study was to evaluate the effect of different combinations of insulin and FSH concentrations in culture media containing GH on the in vitro follicle morphology, antrum formation, growth rates, estradiol (E2) production, oocyte viability and maturation as well as gene expression for FSHR, GHR, INSR, CYP19A1, CYP17, 3ßHSD. Secondary follicles were individually cultured for 18 days in a basic medium containing 50ng/mL GH supplemented with low insulin concentration (INS-LW: 10ng/mL) or high insulin concentration (INS-HG: 10μg/mL) alone or with a fixed FSH concentration (FSH100: 100ng/mL) or with increasing FSH concentrations (FSH-SEQ: 100ng/mL, days 0-6; 500ng/mL, days 6-12; 1000ng/mL days 12-18). In the INS-LW treatment was observed a higher (P<0.05) incidence of normal follicles at day 18 of culture. However, overall higher (P<0.05) follicular growth, oocyte diameter and meiotic resumption rates were obtained using INS-HG+FSH 100. The INS-HG and INS-HG+FSH100 treatments showed higher E2 production and mRNA levels for CYP19A1, CYP17, 3βHSD when compared to INS-LW and INS-LW+FSH100. However, the addition of increasing FSH concentration, regardless of insulin concentration, did not improve the follicular growth, meotic resumption, E2 production or gene expression of steroidogenic enzymes when compared with INS-HG+FSH100. In conclusion, in presence of GH, a basic medium supplemented with 10μg/mL insulin and 100μg/mL FSH throughout the culture period, improves follicular and oocyte growth, oocyte meiotic resumption and E2 production from isolated preantral caprine follicles cultured in vitro.
本研究的目的是评估在含有生长激素（GH）的培养基中，胰岛素和促卵泡激素（FSH）不同浓度组合对体外培养卵泡的形态、腔形成、生长速率、雌二醇（E2）分泌、卵母细胞活力与成熟以及促卵泡激素受体（FSHR）、生长激素受体（GHR）、胰岛素受体（INSR）、细胞色素P450 19A1（CYP19A1）、细胞色素P450 17A1（CYP17）、3β-羟基类固醇脱氢酶（3βHSD）基因表达的影响。次级卵泡在含有50ng/mL GH的基础培养基中单独培养18天，该基础培养基分别补充低胰岛素浓度（INS-LW：10ng/mL）或高胰岛素浓度（INS-HG：10μg/mL），单独使用或与固定的FSH浓度（FSH100：100ng/mL）一起使用，或与递增的FSH浓度（FSH-SEQ：第0 - 6天100ng/mL；第6 - 12天500ng/mL；第12 - 18天1000ng/mL）一起使用。在INS-LW处理组中，培养第18天时正常卵泡的发生率较高（P<0.05）。然而，使用INS-HG + FSH 100时，卵泡总体生长、卵母细胞直径和减数分裂恢复率更高（P<0.05）。与INS-LW和INS-LW + FSH100相比，INS-HG和INS-HG + FSH100处理组的E2分泌以及CYP19A1、CYP17、3βHSD的mRNA水平更高。然而，与INS-HG + FSH100相比，无论胰岛素浓度如何，增加FSH浓度并不能改善卵泡生长、减数分裂恢复、E2分泌或类固醇生成酶的基因表达。总之，在有GH存在的情况下，在整个培养期间补充10μg/mL胰岛素和100μg/mL FSH的基础培养基，可改善体外培养的分离前腔山羊卵泡的卵泡和卵母细胞生长、卵母细胞减数分裂恢复以及E2分泌。
