Vanholder T, Lmr Leroy J, Van Soom A, Maes D, Coryn M, Fiers T, de Kruif A, Opsomer G
Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.
Anim Reprod Sci. 2006 Mar;92(1-2):51-63. doi: 10.1016/j.anireprosci.2005.05.014. Epub 2005 Jun 22.
Elevated serum non-esterified fatty acid (NEFA) levels associated with a negative energy balance (NEB) may affect ovarian function and hence reproductive performance in high-yielding dairy cows. We have investigated the individual and combined effects of the three major NEFAs on bovine theca cell proliferation and steroidogenesis in vitro. Theca cells from healthy large follicles (>8 mm) obtained from slaughterhouse ovaries were cultured in serum free medium in the presence of 0, 50, 150 and 200 microM of palmitic acid (PA; C16:0); 0, 50, 150 and 250 microM of stearic acid (SA; C18:0); and/or 0, 50, 150 and 250 microM of oleic acid (OA; C18:1). Progesterone and androstenedione concentrations were measured in spent medium after 48 h of culture and cell numbers were determined spectrophotometrically per culture well. Cell viability was assessed by annexin-V FITC/propidium iodide staining. Only the treatment with 200 microM of PA inhibited cell proliferation (P<0.001) when tested individually, both of the mixtures tested (M1=100 microM of PA, 130 microM of SA and 140 microM of OA; M2=200 microM PA, 260 microM of SA and 280 microM of OA) reduced cell numbers (P<0.001). Progesterone and androstenedione production, both per well and per 10(4) cells, were not affected by any of the treatments, with the exception of M2. This mixture reduced progesterone production per well and per 10(4) cells (P<0.05). The effects observed were most likely caused by the cytotoxic action of the NEFAs, as demonstrated by the increased percentage of early apoptotic (M1) and late apoptotic/necrotic cells (M1 and M2) in the combination treatments (P<0.05). When combined, elevated physiological concentrations of PA, SA and OA can modulate theca cell proliferation and steroidogenesis in vitro by reducing theca cell viability. These NEFAs may be one of the mediators through which NEB compromises ovarian functioning and thus fertility in high-yielding dairy cows.
与负能量平衡（NEB）相关的血清非酯化脂肪酸（NEFA）水平升高可能会影响高产奶牛的卵巢功能，进而影响其繁殖性能。我们研究了三种主要NEFA对体外培养的牛卵泡膜细胞增殖和类固醇生成的单独及联合作用。从屠宰场卵巢获取的健康大卵泡（>8 mm）中的卵泡膜细胞，在无血清培养基中培养，分别添加0、50、150和200微摩尔的棕榈酸（PA；C16:0）；0、50、150和250微摩尔的硬脂酸（SA；C18:0）；和/或0、50、150和250微摩尔的油酸（OA；C18:1）。培养48小时后，测定培养液中孕酮和雄烯二酮的浓度，并通过分光光度法测定每个培养孔中的细胞数量。通过膜联蛋白-V FITC/碘化丙啶染色评估细胞活力。单独测试时，只有200微摩尔的PA处理抑制细胞增殖（P<0.001），所测试的两种混合物（M1 = 100微摩尔的PA、130微摩尔的SA和140微摩尔的OA；M2 = 200微摩尔的PA、260微摩尔的SA和280微摩尔的OA）均减少了细胞数量（P<0.001）。除M2外，各处理对每个培养孔和每10⁴个细胞的孕酮和雄烯二酮生成均无影响。这种混合物降低了每个培养孔和每10⁴个细胞的孕酮生成（P<0.05）。如联合处理中早期凋亡细胞（M1）和晚期凋亡/坏死细胞（M1和M2）百分比增加所示（P<0.05），观察到的这些影响很可能是由NEFA的细胞毒性作用引起的。当PA、SA和OA以生理浓度联合时，可通过降低卵泡膜细胞活力来调节体外培养的卵泡膜细胞增殖和类固醇生成。这些NEFA可能是NEB损害高产奶牛卵巢功能及生育能力的介质之一。
