Chakravorty A, Mahesh V B, Mills T M
Department of Physiology and Endocrinology, Medical College of Georgia, Augusta 30912.
J Reprod Fertil. 1993 Jan;97(1):91-100. doi: 10.1530/jrf.0.0970091.
Previous studies in this laboratory have demonstrated the presence of a peptide that inhibits granulosa cell proliferation in medium sized follicles. This peptide was produced after 72-96 h of exposure to diethylstilboestrol (DES). This study analyses oestrogen and gonadotrophin modulation of this and another stimulatory peptide found in large follicles. Intact, immature, female rats were assigned to the following study groups: (i) one to four injections of DES (2 mg per rat) given at intervals of 24 h, animals killed 24 h after the last injection; (ii) DES at 0 and 24 h, animals killed 24 or 48 h after the last injection; and (iii) DES plus pentobarbital (37 mg kg-1 body weight) at 30 h, animals killed 48 h after the last injection. Small, medium and large follicles (diameters of < 200, 200-400 and > 400 microns, respectively) were isolated from ovaries, granulosa cells were harvested and follicular fluid supernatant (FFS) was collected. FFS proteins were tested for their effects on incorporation of [3H]thymidine into granulosa cell DNA. Results showed that unfractionated FFS protein (150 micrograms) from medium follicles in groups (i) (three and four injections only), (ii) and (iii) inhibited [3H]thymidine incorporation. Pentobarbital blockage of gonadotrophin secretion had no effect on the inhibitory peptide activity and two injections of diethylstilboestrol were enough to stimulate synthesis of the inhibitory peptide, provided sufficient time was allowed; FFS protein (150 micrograms) from large follicles was stimulatory, but only when it was collected 24 h after the second injection, and the effect was abolished with pentobarbital treatment. Fractionation of pooled FFS into proteins with three molecular mass ranges (< 10 kDa, 10-30 kDa and > 30 kDa) showed that the inhibitory activity was in the < 10 kDa fraction while the > 30 kDa fraction stimulated thymidine incorporation. The number of medium and large follicles increased 24 h after the second DES injection, but the number of granulosa cells in the large follicles was significantly reduced after pentobarbital blockage of gonadotrophins. Taken together, these findings show that (i) the inhibitory peptide is induced in effective amounts in the medium follicles 48 h after the second DES injection and induction is not modulated by gonadotrophins; and (ii) stimulatory activity seen in the large follicles is transient and under gonadotrophic control, since blockage of follicle-stimulating hormone (FSH) secretion led to loss of stimulatory activity and a resulting reduction in number of granulosa cells in large follicles.(ABSTRACT TRUNCATED AT 400 WORDS)
本实验室之前的研究已证明存在一种可抑制中等大小卵泡中颗粒细胞增殖的肽。该肽是在暴露于己烯雌酚（DES）72 - 96小时后产生的。本研究分析雌激素和促性腺激素对这种肽以及在大卵泡中发现的另一种刺激性肽的调节作用。将完整的未成熟雌性大鼠分为以下研究组：（i）每隔24小时注射一至四次DES（每只大鼠2毫克），在最后一次注射后24小时处死动物；（ii）在0和24小时注射DES，在最后一次注射后24或48小时处死动物；（iii）在30小时注射DES加戊巴比妥（37毫克/千克体重），在最后一次注射后48小时处死动物。从卵巢中分离出小、中、大卵泡（直径分别<200、200 - 400和>400微米），收获颗粒细胞并收集卵泡液上清液（FFS）。检测FFS蛋白对[3H]胸腺嘧啶掺入颗粒细胞DNA的影响。结果显示，（i）组（仅三次和四次注射）、（ii）组和（iii）组中来自中等卵泡的未分级FFS蛋白（150微克）抑制了[3H]胸腺嘧啶的掺入。戊巴比妥阻断促性腺激素分泌对抑制肽活性无影响，并且两次注射己烯雌酚足以刺激抑制肽的合成，前提是给予足够的时间；来自大卵泡的FFS蛋白（150微克）具有刺激性，但仅在第二次注射后24小时收集时才有此作用，并且戊巴比妥处理可消除该作用。将合并的FFS分离为三个分子量范围（<10 kDa、10 - 30 kDa和>30 kDa）的蛋白质，结果显示抑制活性存在于<10 kDa的部分，而>30 kDa的部分刺激胸腺嘧啶掺入。第二次DES注射后24小时，中等和大卵泡的数量增加，但在戊巴比妥阻断促性腺激素后，大卵泡中颗粒细胞的数量显著减少。综上所述，这些发现表明：（i）第二次DES注射后48小时，中等卵泡中可有效诱导出抑制肽，且诱导不受促性腺激素调节；（ii）大卵泡中观察到的刺激活性是短暂的且受促性腺激素控制，因为阻断促卵泡激素（FSH）分泌会导致刺激活性丧失，进而使大卵泡中颗粒细胞数量减少。（摘要截断于400字）
