Days Of E2 Research Articles

Progesterone-Dependent Cathepsin L Proteolytic Activity in Cat Uterine Flushings1

Sequence analysis of a cDNA clone for the progesterone-dependent protein (PDP) of the cat uterus revealed that PDP may be cathepsin L. This study was undertaken to directly measure the cathepsin L activity in uterine flushings from pregnant and ovariectomized steroid-treated animals in order to confirm that PDP is cathepsin L. Optimum activity toward the substrate Z-Phe-Arg-NMec was observed at a pH of 5-6. Z-Phe-Phe-CHN2, a specific inhibitor of cathepsin L, significantly inhibited the proteolytic activity present in uterine flushings. Immunoabsorption of PDP from uterine flushings obtained from progesterone (P)-treated cats reduced cathepsin L proteolytic activity to levels observed in ovariectomized and estradiol (E2)-treated animals. In E2-primed and E2 + P-treated animals, proteolytic activity in uterine flushings was detectable after 7 days and peaked after 11-13 days of E2 + P treatment. This proteolytic activity was also dramatically increased before implantation (10-12 days after coitus) in pregnant cats. Thus, our data indicate that changes in cathepsin L activity in uterine flushings are correlated with changes in PDP, the uterine protein synthesized and released from the epithelial cells of the deep uterine glands. PDP, via its cathepsin L proteolytic activity, may play a role in the implantation process.

Tissue-specific regulation of rat estrogen receptor mRNAs.

The estrogen receptor (ER) is present in a wide variety of mammalian tissues and is required for physiological estrogen responses, including estrogen-induced tissue-specific changes in gene expression. We studied the estrogen regulation of the mRNAs encoding the ER in rat uterus, liver, and pituitary. Ovariectomized (21-28 day post surgery) female CD-1 rats were injected daily with 17 beta-estradiol (E2, 10 micrograms/100 g BW) for 0, 1, or 4 h, 1, 3, or 7 days and compared with intact controls. Steady-state levels of ER mRNA were quantified using a human ER cDNA probe. Only one hybridizing species of approximately 6.2 kilobase (kb) was detected in uterine and liver RNA, similar to that observed in MCF7 human breast cancer cells. However, the ER mRNA regulation by E2 differed in direction depending on the tissue examined. In uterus, ER mRNA increased 3- to 6-fold after ovariectomy, and returned to intact levels within 24 h of E2 replacement. In contrast, liver ER mRNA declined 1.5- to 3-fold after ovariectomy and returned to intact levels after 1-3 days of E2. In pituitary tissue two hybridizing forms of ER mRNA were observed, with one species migrating at 6.2 kb, equivalent to the form in other tissues, and a second smaller species at approximately 5.5 kb. The lower molecular weight species varied somewhat in abundance from animal to animal, averaging about 20% of the intensity of the 6.2 kb band. The ER mRNA forms were regulated positively with E2.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical localization of estrogen receptors in the macaque endometrium during the luteal-follicular transition.

We examined changes in estrogen receptors (ERs) in endometrial stromal and epithelial cells in cynomolgus macaques during artificially induced menstruation and repair. We used Silastic implants filled with either estradiol (E2) or progesterone (P) to treat spayed animals for 14 days with E2 followed by 14 days of E2 plus P. We then withdrew the P (but not the E2) implants and removed uteri 0, 0.5, 1, 2, 3, 4, 5, 7, and 14 days later. Uterine tissues were assayed biochemically for ER content, fixed for histology and frozen for immunocytochemistry of ER with monoclonal antiestrophilins. On day 0, ER levels in endometrium were low [1330 +/- 201 (n = 9) fmol/mg DNA]. An increase in total receptor was evident by 3-4 days of P withdrawal 2762 +/- 190 (n = 6) fmol/mg DNA; P less than 0.001]. Total receptor concentrations increased linearly with time from 0.5-7 days of P withdrawal (r = 0.88). On day 0, staining for nuclear ER in the glandular epithelium and stroma of zones I, II, and III of the endometrium was negative. Beginning at 12-24 h and continuing through 4 days of P withdrawal, nuclear staining became detectable and increased in intensity only in endometrial stromal fibroblasts and myometrial smooth muscle cells. The glandular epithelium of the endometrium did not develop nuclear staining until 5-7 days of P withdrawal, coincident with a 10-fold increase in the mitotic index of the epithelium of the upper zones. Thus, the increase in endometrial ER levels that occurred during the first 5 days of an induced luteal-follicular transition took place almost exclusively in stromal fibroblasts.

Direct arterial vascularization of estrogen-induced prolactin-secreting anterior pituitary tumors.

The rat anterior pituitary gland (AP) receives all of its blood supply via the hypophyseal portal circulation. We now report, in rats with estradiol (E2)-induced prolactin-secreting tumors, that newly formed arteries directly supply the AP and that this arteriogenesis is closely correlated with the sensitivity of two strains of rats to the tumorigenic action of E2. Fischer 344 rats, a strain extremely sensitive to E2, and Sprague-Dawley rats, a less sensitive strain, were ovariectomized and implanted with E2-filled or empty Silastic capsules. Ten to 63 days later, microspheres (15 microns) were injected into the heart. Normally microspheres do not reach the AP because they are trapped in the primary portal capillary plexus. Some animals were also perfused with vascular cast material. In Fischer rats, after 63 days of E2, the pituitary weight, serum prolactin, and number of microspheres in the AP were 5-, 42-, and 18-fold greater than control values, respectively. The same parameters in E2-treated Sprague-Dawley rats were 2-, 27-, and 7-fold greater than control values. Vascular casts from E2-treated Fischer rats revealed numerous arteries entering the AP. No arteries to the AP were observed in Sprague-Dawley controls. These results show that E2-induced tumorigenesis of the AP is associated with the development of a direct arterial blood supply. We hypothesize that the regions supplied by these new arteries would receive systemic blood containing subphysiological concentrations of dopamine. The loss of dopaminergic inhibition in concert with E2 stimulation may lead to tumor formation.

Uterine responses to estradiol in the neonatal rat.

Although single doses of estrogens are known to be ineffective in stimulating complete uterine responses in newborn rats, repeated doses elicit toxic responses that become evident in adulthood. In this study, neonates injected daily from birth with 10 micrograms 17 beta-estradiol (E2) demonstrated significant uterine wet weight gain by day 3 and near-maximum growth (230% of control) by day 5. Elevated uterine weight can be maintained by repeated daily injections at least through day 13. Other uterine growth responses after 5 days of E2 (as percent of control) are: dry weight, 163%; DNA content, 193%; protein content, 211%; and nuclear estrogen receptor, 890%. Ornithine decarboxylase activity increased to 520% of control when measured 6 h after a single E2 injection on day 5. Histological examination of uteri from animals treated for 5 days reveals an altered stroma with evidence of circular muscle differentiation, while the lumenal epithelium, which is cuboidal in controls, becomes columnar after E2 injections. These data suggest that estrogens act in fundamentally the same manner in the neonatal uterus as in the adult uterus, although the appearance of the complete response appears to e slower in the neonate. The estrogen-induced precocious development we describe suggests that an estrogen may be involved in the normal postnatal development of uterine estrogen responsiveness. Adult toxicity, resulting from repeated neonatal estrogen dosing, may partly be a consequence of continual hormone action inducing developmentally inappropriate responses.

Effects of Estrogen on Primary Ovine Pituitary Cell Cultures: Stimulation of Prolactin Secretion, Synthesis, and Preprolactin Messenger Ribonucleic Acid Activity*

Estrogen regulation of PRL synthesis and secretion was studied in primary ovine pituitary cell cultures. Medium PRL, [3H]leucine incorporation into PRL, and pre-PRL messenger RNA (mRNA) activity were determined in cultures incubated in control or 17β-estradiol (E2)-containing medium. We consistently observed a significant increase in medium PRL after 2 days of E2 treatment. A maximal response of 150–200% of control was reached after 6–8 days of E2. The response increased with increasing doses of E2 in the physiological range (10-9-10-8 M) and was maximal at about 10-9 M E2. Sodium dodecyl sulfate gel electrophoresis of the soluble proteins labeled by a 1-h incubation in radioactive leucine showed that the synthesis of only one major protein band which comigrated with authentic ovine PRL was stimulated. Immunoprecipitation of the labeled cell homogenates confirmed that PRL synthesis was increased. Five days of E2 increased medium PRL to 150% of controls, PRL synthesis to 183% of controls, and pre-PRL mRNA ...