Attempts to establish a screening procedure for endocrine disrupters by ovulation analysis using the intrabursal injection method

本研究的目的是建立一种利用哺乳动物卵泡内注射分析内分泌干扰物的筛选方法的基础。我们首先尝试寻找适合这种方法的分子标记。为此，我们检测了缓激肽B2受体和新型基质金属蛋白酶MMP-23是否适合作为标记物。在猪和小鼠卵巢中均检测到B2受体mRNA和受体蛋白。进一步详细的形态学检查发现，B2受体mRNA在卵巢所有生长卵泡颗粒细胞中均有组成性表达，且mRNA水平在整个排卵期没有变化。这些发现表明B2受体可能不是一个合适的标记。用大鼠卵巢检测MMP-23的表达。在卵泡生长早期，mmp - 23mrna定位于生长卵泡颗粒细胞，但在完全发育的卵泡中，其表达转移到外鞘层。基于这些结果，我们推测MMP-23可能是卵泡生长的标志而不是排卵的标志。应用雌性大鼠和小鼠的排卵分析方法，研究了两种已知的内分泌干扰物DES和DEHP对排卵过程的影响。大鼠手背内注射DES和DEHP未导致排卵卵数明显减少。然而，经des处理的母鼠的雌性后代排卵数量明显减少。这些结果表明，DES处理对后代的生殖能力产生了深远的影响。

he aim of this study was to establish the basis for a acreening method of endocrine disrupters using intrabursal injection analysis of mammalian ovulation.We first attempted to search for molecular markers suitable for this method. To this end, bradykinin B2 receptor and MMP-23(a new type of matrix metalloproteinase)were examined whether they would be appropriate as markers. The B2 receptor mRNA and receptor protein were both detected in the ovaries of pig and mouse. Further detailed morphological examination revealed that the B2 receptor mRNA was constitutively expressed in the granulosa cells of all growing follicles in the ovary, and that the mRNA levels did not change through the periovulatory period. These findings indicate that the B2 receptor may not serve as an appropriate marker. The expression of MMP-23 was examined using rat ovaries for the same purpose. MMP-23 mRNA was localized at granulosa cells of growing follicles in the early stages of follicle growth, but its expression was shifted to the outer thecal layer of fully grown follicles. Based on these results, we presumed that MMP-23 could be a marker for follicle growth rather than for ovulation.Effects of two known endocrine disrupters, DES and DEHP, on ovulatory process were examined by applying for the ovulation analysis method using female rats and mice. Intrabursal injection of DES and DEHP into rats did not resulted in significant decrease in the numbers of ovulated eggs. However, female offsprings of the DES-treated mother mice ovulated substantially reduced numbers of eggs. These results indicate that DES treatment affected profoundly reproductive ability of the offspring.