Martoriati A, Caillaud M, Goudet G, Gérard N
Equipe Reproduction Equine, Unité de Physiologie de la Reproduction et des Comportements, INRA 37380 Nouzilly, France.
Reproduction. 2003 Oct;126(4):509-15. doi: 10.1530/rep.0.1260509.
Interleukin 1 beta (IL-1 beta) inhibits the LH-induced resumption of meiosis of equine oocytes in vitro. The present study was performed to clarify this inhibitory effect of IL-1 beta by testing increasing concentrations of IL-1 beta, and by measuring the effect of addition of IL-1 receptor antagonist (IL-1RA) to the culture medium. The effect of IL-1 beta on epidermal growth factor (EGF)-induced resumption of meiosis was also studied. Cumulus-oocyte complexes (COCs) were collected from subordinate follicles on ovaries obtained from an abattoir. In five distinct experiments, COCs were cultured for 30 h and nuclear maturation of oocytes was evaluated by DNA staining. In Expt 1, seven different media were tested: medium 1 (TCM199+BSA); medium 2 (medium 1+50 ng IL-1 beta ml(-1)); medium 3 (medium 1+eLH); and media 4, 5, 6 and 7 (medium 3 containing 0.1, 1.0, 10.0 and 50.0 ng IL-1 beta ml(-1), respectively). In Expt 2, four different media were tested: medium 1 (TCM199+BSA+eLH); medium 2 (medium 1+50 ng IL-1 beta ml(-1)); and media 3 and 4 (medium 2+IL-1RA at 50 and 100 ng ml(-1), respectively). In Expt 3, three different media were tested: medium 1 (TCM199+BSA+eLH); medium 2 (medium 1+50 ng IL-1RA ml(-1)); and medium 3 (medium 2+50 ng IL-1 beta ml(-1)). In Expt 4, four different media were tested: medium 1 (TCM199+BSA+eLH); and media 2, 3 and 4 (medium 1+IL-1RA at 50, 100 and 150 ng ml(-1), respectively). In Expt 5, three different media were tested: medium 1 (TCM199+BSA+EGF); medium 2 (medium 1+50 ng IL-1 beta ml(-1)); and medium 3 (medium 2+50 ng IL-1RA ml(-1)). In Expt 1, LH alone induced an increase in the rate of in vitro maturation (IVM) of equine oocytes (P<0.05), whereas IL-1 beta alone did not have any effect compared with medium 1. IL-1 beta (50 ng ml(-1)) significantly inhibited the eLH-induced IVM of oocytes (P<0.05) compared with medium 3. A decrease in rate of maturation was observed from a concentration of 10 ng IL-1 beta ml(-1) onwards. In Expt 2, the presence of IL-1RA in the culture medium inhibited the effect of IL-1 beta and restored the rate of oocyte maturation (P<0.05) observed in the presence of LH alone. In Expts 3 and 4 it was demonstrated that IL-1RA alone had no positive effect on the eLH-induced rate of maturation. In Expt 5, IL-1 beta inhibited the EGF-induced resumption of meiosis (P<0.05). The addition of IL-1RA inhibited this effect and restored the rate of oocyte maturation (P<0.05) observed with EGF alone. In conclusion, the present data confirm the inhibitory effect of IL-1 beta on IVM of equine oocytes induced by eLH and demonstrate its inhibitory effect on EGF-induced oocyte maturation. The rate of maturation decreased in a dose-dependent way and the lowest rate of maturation was observed at 50 ng IL-1 beta ml(-1) (P<0.05). The use of IL-1RA inhibited these effects, demonstrating that the action of IL-1 beta is receptor-mediated. Moreover, the results clearly show that, in equine species, IL-1 beta is involved in the physiology of COCs by regulating resumption of meiosis.
白细胞介素1β（IL-1β）在体外可抑制促黄体生成素（LH）诱导的马属动物卵母细胞减数分裂的恢复。本研究旨在通过检测不同浓度的IL-1β，并测定向培养基中添加IL-1受体拮抗剂（IL-1RA）的效果，来阐明IL-1β的这种抑制作用。同时还研究了IL-1β对表皮生长因子（EGF）诱导的减数分裂恢复的影响。从屠宰场获取的卵巢上的次级卵泡中收集卵丘-卵母细胞复合体（COCs）。在五个不同的实验中，将COCs培养30小时，并通过DNA染色评估卵母细胞的核成熟情况。在实验1中，测试了七种不同的培养基：培养基1（TCM199 + BSA）；培养基2（培养基1 + 50 ng IL-1β ml⁻¹）；培养基3（培养基1 + eLH）；以及培养基4、5、6和7（培养基3分别含有0.1、1.0、10.0和50.0 ng IL-1β ml⁻¹）。在实验2中，测试了四种不同的培养基：培养基1（TCM199 + BSA + eLH）；培养基2（培养基1 + 50 ng IL-1β ml⁻¹）；以及培养基3和4（培养基2分别添加50和100 ng ml⁻¹的IL-1RA）。在实验3中，测试了三种不同的培养基：培养基1（TCM199 + BSA + eLH）；培养基2（培养基1 + 50 ng IL-1RA ml⁻¹）；以及培养基3（培养基2 + 50 ng IL-1β ml⁻¹）。在实验4中，测试了四种不同的培养基：培养基1（TCM199 + BSA + eLH）；以及培养基2、3和4（培养基1分别添加50、100和150 ng ml⁻¹的IL-1RA）。在实验5中，测试了三种不同的培养基：培养基1（TCM199 + BSA + EGF）；培养基2（培养基1 + 50 ng IL-1β ml⁻¹）；以及培养基3（培养基2 + 50 ng IL-1RA ml⁻¹）。在实验1中，单独使用LH可诱导马属动物卵母细胞的体外成熟（IVM）率增加（P<0.05），而单独使用IL-1β与培养基1相比没有任何影响。与培养基3相比，IL-1β（50 ng ml⁻¹）显著抑制了eLH诱导的卵母细胞IVM（P<0.05）。从10 ng IL-1β ml⁻¹起观察到成熟率下降。在实验2中，培养基中存在IL-1RA可抑制IL-1β的作用，并恢复了仅在LH存在时观察到 的卵母细胞成熟率（P<0.05）。在实验3和4中表明，单独使用IL-1RA对eLH诱导的成熟率没有积极影响。在实验5中，IL-1β抑制了EGF诱导的减数分裂恢复（P<0.05）。添加IL-1RA可抑制这种作用，并恢复了仅用EGF时观察到的卵母细胞成熟率（P<0.05）。总之，目前的数据证实了IL-1β对eLH诱导的马属动物卵母细胞IVM的抑制作用，并证明了其对EGF诱导的卵母细胞成熟的抑制作用。成熟率呈剂量依赖性下降，在50 ng IL-1β ml⁻¹时观察到最低成熟率（P<0.05）。使用IL-1RA可抑制这些作用，表明IL-1β的作用是受体介导的。此外，结果清楚地表明，在马属动物中，IL-1β通过调节减数分裂的恢复参与了COCs的生理过程。
