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

本研究的目的是通过对哺乳动物排卵的腔内注射分析，为建立一种内分泌干扰物的筛选方法奠定基础。为此，研究了缓激肽B2受体和基质金属蛋白酶-23(一种新型的基质金属蛋白酶)是否适合作为标记物。在猪和小鼠卵巢中均检测到B2受体基因和受体蛋白的表达。进一步详细的形态观察表明，B2受体mRNA在卵巢内所有生长卵泡的颗粒细胞中都有结构性表达，并且在整个排卵期内其表达水平没有变化。这些发现表明，B2受体可能不能作为一个合适的标记物。为了同样的目的，我们用大鼠卵巢检测了基质金属蛋白酶-23的表达。在卵泡发育的早期，基质金属蛋白酶-23的表达定位于生长卵泡的颗粒细胞，但在发育成熟的卵泡中，其表达转移到了卵泡膜外。基于这些结果，我们推测，基质金属蛋白酶-23可能是一个卵泡生长而不是排卵的标志。应用排卵分析方法，以雌性大鼠和小鼠为实验对象，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。大鼠囊内注射DES和DEHP后，排卵量未见明显减少。然而，DES处理的母鼠的雌性后代排卵大大减少了卵子的数量。这些结果表明，DES处理对后代的繁殖力有很大的影响。

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.