Study on uterine ribonucleic acid with estrogenic activity

The influence of intrauterine instillation of uterine RNA on protein, lipid and RNA synthesis in uteri of ovariectomized rats was studied. Extensive purification of the uterine RNA was performed to exclude any residual extradiol-17β in the preparations. RNA was isolated from the uteri of estrogen-stimulated rats by phenol fractionation or diethyloxydifonnate treatment and washed twice with 95 per cent ethanol. The RNA preparations (8–10mg) were dissolved in 20 ml of saline and tested for estrogen-like activity by a single instillation into ligated uterine horns of ovariectomized rats. [3H]uridine (100μCi) and [14C]glycine (25 μCi) were administered intravenously to ovariectomized rats at various times after the intrauterine instillation of RNA. Rats were killed 30 min after the administration of uridine and 30 or 60 min after glycine. The incorporation of radioactivity into total RNA, lipids and proteins was determined. Manipulative procedures such as ligation of uterine horns and/or intrauterine instillation of saline stimulated uterine RNA synthesis. When uterine and liver RNA obtained from estrogenstimulated rats and washed with ethanol but not with ether were administered, however, the increase in RNA synthesis was significantly greater than the procedural control values at 4 and 8 h after the intrauterine instillation. To exclude any residual estrogen the uterine RNA preparation was extracted with ether 6 times. When the sediment from the organic phase was dissolved in oil and assayed, it stimulated uterine RNA synthesis. The uterine RNA obtained from control non-stimulated ovariectomized rats stimulated RNA synthesis. However, the values obtained were equivalent to the ones observed with saline instillation or following ligation of the uterine horns. Uterine lipid synthesis was increased, also, by ligation or intrauterine instillation of saline. The increase in lipid synthesis initiated by uterine RNA was equivalent to the procedural control values. Protein synthesis, however, was increased by uterine RNA obtained after ethanol washing and the sediment of the ether phase. To analyze for the possible presence of residual estradiol-17β in the uterine RNA preparations, the contents of estradiol-17β in the preparations were determined.[3H] or [14C]estradiol-17β (10 μg) was administered intravenously to ovariectomized rats. The RNA were prepared from the uterus, kidney and liver 4 h after the hormone treatment. The content of estradiol-17β in the RNA preparations as calculated from the radioactivity ranged from 0.05 to 3.5 pg per 10 μg of RNA. It was determined that a single dose of 1 pg of estradiol-17β administered by intrauterine instillation stimulated RNA synthesis at 4h, increased slightly lipid synthesis at 30 min and accelerated protein synthesis at 4 h. The present results reveal that uterine and liver RNA extracted with phenol and washed with ethanol but not with ether contains small amounts of estrogen which may account for the stimulatory action of such extracts. After repeated washings of RNA with ether the estrogen content was reduced to less than 10−2pg of estradiol-17β per 10μg RNA. The ether-extracted uterine or liver RNA did not stimulate RNA synthesis, but did increase protein synthesis at 24 and 48 h after instillation. RNA extracted from estrogen-free proliferative uteri induced by intra-uterine pressure possesses similar biologic activity. These results indicate that the ability to stimulate protein synthesis may be an intrinsic property of RNA from proliferating uteri.