Chakravorty A, Mahesh V B, Mills T M
Department of Physiology & Endocrinology, Medical College of Georgia, Augusta 30912.
J Reprod Fertil. 1991 Jul;92(2):323-32. doi: 10.1530/jrf.0.0920323.
Intact immature female rats were treated with 1, 2, 3 or 4 subcutaneous injections of 2 mg diethylstilboestrol (DES)/rat at intervals of 24 h and then killed. Ovaries were collected, cleaned, enzymically digested and serially filtered through Teflon sieves to yield follicles of diameter less than 200 microns (small), 200-400 microns (medium) and greater than 400 microns (large). Follicular supernatant was collected and granulosa cells were extracted from these isolated follicles. There was a general increase in [3H]thymidine incorporation in all sizes of follicles after 1 or 2 DES injections, the increase in the medium and large follicles being significant after 2 doses. With 3 and 4 injections of DES, there was a sudden decrease in the rates of [3H]thymidine incorporation, particularly in the medium-sized follicles, which also had higher concentrations of follicular supernatant protein. Protein contents in small and large follicles did not change significantly. The follicular supernatant protein had a specific and dose-dependent inhibitory effect on [3H]thymidine incorporation when added to cultures of rapidly dividing granulosa cells. Addition of the same amounts of bovine serum albumin (BSA) to the cultures had no effect. Heat-denaturing did not abolish the inhibition by the protein. Removal of the protein from the cultures after the first 48 h resulted in a rebound increase in [3H]thymidine incorporation during the following 48 h, showing that the inhibitory effects were reversible. Though aromatase activity after 1 or 2 DES injections abruptly decreased after 3 and 4 injections, follicular supernatant protein had no effect on steroidogenesis in cultured granulosa cells. Taken together, these findings suggest that oestrogen can inhibit follicular development, depending on the duration of exposure. We propose that the inhibitory effects of DES on cell proliferation are mediated via the synthesis of a specific peptide factor which is produced in high amounts in the medium-sized follicles only, on prolonged exposure to the oestrogen. This factor may be autocrine or paracrine, serving as an in-built autoregulatory control mechanism for follicle development, particularly at pro-oestrus, when oestrogen concentrations are highest.
选用未成熟的雌性大鼠，每隔24小时皮下注射1、2、3或4次，每次剂量为2mg己烯雌酚（DES）/只大鼠，随后将其处死。收集卵巢，清理后进行酶消化，并依次通过聚四氟乙烯筛网过滤，以获得直径小于200微米（小）、200 - 400微米（中）和大于400微米（大）的卵泡。收集卵泡上清液，并从这些分离出的卵泡中提取颗粒细胞。注射1或2次DES后，所有大小卵泡中[³H]胸腺嘧啶核苷掺入量普遍增加，注射2次后，中、大卵泡中的增加量显著。注射3次和4次DES后，[³H]胸腺嘧啶核苷掺入率突然下降，尤其是在中等大小的卵泡中，这些卵泡的卵泡上清液蛋白浓度也较高。小卵泡和大卵泡中的蛋白含量没有显著变化。当将卵泡上清液蛋白添加到快速分裂的颗粒细胞培养物中时，对[³H]胸腺嘧啶核苷掺入具有特异性和剂量依赖性抑制作用。向培养物中添加相同量的牛血清白蛋白（BSA）则没有效果。加热变性并未消除该蛋白的抑制作用。在最初的48小时后从培养物中去除该蛋白，导致在接下来的48小时内[³H]胸腺嘧啶核苷掺入量出现反弹增加，表明抑制作用是可逆的。尽管注射1或2次DES后芳香化酶活性在注射3次和4次后突然下降，但卵泡上清液蛋白对培养的颗粒细胞中的类固醇生成没有影响。综上所述，这些发现表明雌激素可抑制卵泡发育，这取决于暴露持续时间。我们推测，DES对细胞增殖的抑制作用是通过一种特定肽因子的合成介导的，只有在长时间暴露于雌激素的情况下，该因子才会在中等大小的卵泡中大量产生。该因子可能是自分泌或旁分泌的，作为卵泡发育的一种内在自动调节控制机制，特别是在雌激素浓度最高的发情前期。
