Pomerantz S H, Bilello P A
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
Gamete Res. 1987 Jul;17(3):267-78. doi: 10.1002/mrd.1120170310.
We describe an assay for oocyte maturation inhibitor (OMI) using the progesterone-mediated maturation of Xenopus oocytes. The test fraction used was a partially purified fraction from pig follicular fluid, which gave consistent inhibition of maturation in the pig oocyte assay. In the toad assay, oocytes (30-50) from each toad were pretested to determine whether satisfactory maturation was achieved because widespread animal variation was observed. Toads whose oocytes showed greater than 60% maturation in the pretest could be used directly. Toads whose oocytes showed less than 60% maturation were injected with pregnant mares serum gonadotropin (PMSG) in order to increase progesterone-mediated maturation. The dose and time after injection of PMSG before harvesting oocytes, dose and duration of progesterone exposure, and order of exposure of oocytes to OMI and progesterone were important variables. Opposing effects of OMI and progesterone were seen in oocytes from toads receiving 60 IU PMSG. In the routine assay we use animals whose oocytes show greater than 60% maturation in the pretest or animals treated with 12 IU of PMSG 4 to 7 days before use. Oocytes are exposed to the OMI fraction for 1 hr, progesterone is added, and incubations continued until controls reach maximum maturation (5 to 8 hr). The inhibition of toad oocyte maturation by OMI is reversible. The toad and mammalian oocyte assays were compared using more highly purified fractions of OMI and gave identical results.
我们描述了一种利用非洲爪蟾卵母细胞孕酮介导的成熟过程来检测卵母细胞成熟抑制剂（OMI）的方法。所用的测试组分是猪卵泡液的部分纯化组分，在猪卵母细胞检测中它能持续抑制成熟过程。在蟾蜍检测中，对每只蟾蜍的30 - 50个卵母细胞进行预测试，以确定是否实现了令人满意的成熟，因为观察到存在广泛的动物个体差异。预测试中卵母细胞成熟率大于60%的蟾蜍可直接使用。预测试中卵母细胞成熟率小于60%的蟾蜍注射孕马血清促性腺激素（PMSG），以增加孕酮介导的成熟过程。收获卵母细胞前注射PMSG的剂量和时间、孕酮暴露的剂量和持续时间，以及卵母细胞暴露于OMI和孕酮的顺序都是重要变量。在接受60 IU PMSG的蟾蜍的卵母细胞中观察到了OMI和孕酮的相反作用。在常规检测中，我们使用预测试中卵母细胞成熟率大于60%的动物，或在使用前4至7天用12 IU PMSG处理的动物。将卵母细胞暴露于OMI组分1小时，加入孕酮，然后继续孵育，直到对照达到最大成熟度（5至8小时）。OMI对蟾蜍卵母细胞成熟的抑制作用是可逆的。使用更高纯度的OMI组分对蟾蜍和哺乳动物卵母细胞检测进行了比较，结果相同。
