UMR PRC, CNRS, IFCE, INRA, Université de Tours, 37380, Nouzilly, France.
INRA Centre Val de Loire, Physiologie de la Reproduction et des Comportements, 37380, Nouzilly, France.
Reprod Biol Endocrinol. 2018 Apr 26;16(1):40. doi: 10.1186/s12958-018-0357-7.
Docosahexaenoic acid (DHA) is a n-3 polyunsaturated fatty acid (PUFA) belonging to a family of biologically active fatty acids (FA), which are known to have numerous health benefits. N-3 PUFAs affect reproduction in cattle, and notably directly affect follicular cells. In terms of reproduction in cattle, n-3 PUFA-enriched diets lead to increased follicle size or numbers.
The objective of the present study was to analyze the effects of DHA (1, 10, 20 and 50 μM) on proliferation and steroidogenesis (parametric and/or non parametric (permutational) ANOVA) of bovine granulosa cells in vitro and mechanisms of action through protein expression (Kruskal-Wallis) and signaling pathways (non parametric ANOVA) and to investigate whether DHA could exert part of its action through the free fatty acid receptor 4 (FFAR4).
DHA (10 and 50 μM) increased granulosa cell proliferation and DHA 10 μM led to a corresponding increase in proliferating cell nuclear antigen (PCNA) expression level. DHA also increased progesterone secretion at 1, 20 and 50 μM, and estradiol secretion at 1, 10 and 20 μM. Consistent increases in protein levels were also reported for the steroidogenic enzymes, cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1), and of the cholesterol transporter steroidogenic acute regulatory protein (StAR), which are necessary for production of progesterone or androstenedione. FFAR4 was expressed in all cellular types of bovine ovarian follicles, and in granulosa cells it was localized close to the cellular membrane. TUG-891 treatment (1 and 50 μM), a FFAR4 agonist, increased granulosa cell proliferation and MAPK14 phosphorylation in a similar way to that observed with DHA treatment. However, TUG-891 treatment (1, 10 and 50 μM) showed no effect on progesterone or estradiol secretion.
These data show that DHA stimulated proliferation and steroidogenesis of bovine granulosa cells and led to MAPK14 phosphorylation. FFAR4 involvement in DHA effects requires further investigation, even if our data might suggest FFAR4 role in DHA effects on granulosa cell proliferation. Other mechanisms of DHA action should be investigated as the steroidogenic effects seemed to be independent of FFAR4 activation.
二十二碳六烯酸（DHA）是一种 n-3 多不饱和脂肪酸（PUFA），属于具有多种健康益处的生物活性脂肪酸（FA）家族。n-3 PUFAs 会影响牛的繁殖，特别是直接影响卵泡细胞。就牛的繁殖而言，富含 n-3 PUFA 的饮食会导致卵泡大小或数量增加。
本研究的目的是分析 DHA（1、10、20 和 50 μM）对体外牛颗粒细胞增殖和类固醇生成（参数和/或非参数（置换）方差分析）的影响，以及通过蛋白质表达（Kruskal-Wallis）和信号通路（非参数方差分析）分析作用机制，并研究 DHA 是否可以通过游离脂肪酸受体 4（FFAR4）发挥部分作用。
DHA（10 和 50 μM）增加了颗粒细胞的增殖，DHA 10 μM 导致增殖细胞核抗原（PCNA）表达水平相应增加。DHA 还分别在 1、20 和 50 μM 时增加了孕激素的分泌，在 1、10 和 20 μM 时增加了雌二醇的分泌。还报告了类固醇生成酶细胞色素 P450 家族 11 亚家族 A 成员 1（CYP11A1）和羟基-δ-5-类固醇脱氢酶、3β-和类固醇δ-异构酶 1（HSD3B1）以及胆固醇转运蛋白类固醇急性调节蛋白（StAR）的蛋白水平的一致增加，这些都是产生孕激素或雄烯二酮所必需的。FFAR4 在牛卵巢卵泡的所有细胞类型中均有表达，在颗粒细胞中，它靠近细胞膜定位。TUG-891 处理（1 和 50 μM），一种 FFAR4 激动剂，以类似于 DHA 处理的方式增加颗粒细胞增殖和 MAPK14 磷酸化。然而，TUG-891 处理（1、10 和 50 μM）对孕激素或雌二醇的分泌没有影响。
这些数据表明，DHA 刺激了牛颗粒细胞的增殖和类固醇生成，并导致了 MAPK14 的磷酸化。FFAR4 参与 DHA 作用需要进一步研究，即使我们的数据可能表明 FFAR4 在 DHA 对颗粒细胞增殖的作用中的作用。应该研究 DHA 作用的其他机制，因为甾体生成作用似乎独立于 FFAR4 的激活。
