Estrogen induces insulin-like growth factor-I expression in the rat uterus

The inability to convincingly demonstrate a mitogenic effect of estrogen on isolated uterine cells in culture suggests that autocrine or paracrine growth factors may be important in the estrogen-induced uterine proliferative response. Here we report that uterine expression of insulin-like growth factor-I (IGF-I), an important mediator of GH action, is increased after 17 beta-estradiol (5 micrograms/100 g bw, ip) administration to ovariectomized prepubertal rats. An increase in uterine IGF-I mRNA abundance, approximately 14-fold above untreated controls, was apparent 6 h after estrogen administration and the level achieved exceeded that seen in the uterus from intact mature rats during diestrus. In contrast to the increase in IGF-I expression in the uterus, no significant change in serum IGF-I concentration or hepatic or renal IGF-I mRNA abundance was demonstrable after 17 beta-estradiol injection of ovariectomized prepubertal rats. The increase in uterine IGF-I expression, was similar in both pituitary-intact and hypophysectomized, ovariectomized rats. We believe this is the first report of induction of IGF-I expression by estrogen in vivo. As such, the finding expands the role and significance of IGF-I as a mediator of growth beyond that related to GH.     

Activation of 'immediate-early' genes by estrogen is not sufficient to achieve stimulation of DNA synthesis in rat uterus

17 beta-estradiol, a long acting estrogen that is mitogenic for rat uterus in vivo, or the short acting estrogens estriol and 16 alpha-estradiol, not mitogenic on their own, were injected into adult, castrated rats and their effect on uterine gene expression and rate of DNA synthesis were compared. All three compounds increased steady-state mRNA concentration of c-fos, c-jun and c-myc proto-oncogenes to comparable levels (2 hrs after treatment), whereas only 17 beta-estradiol was found to stimulate significantly DNA synthesis (20-22 hrs later). Based on the different retention time of the tested estrogens in rat tissues, it is concluded that a short exposure to the hormone is sufficient to render uterine cells competent to progress through the cell cycle, via activation of 'immediate-early' genes expression, but that stimulation of DNA synthesis requires further changes, achieved via a prolonged exposure of the cells to the estrogenic stimulus.     

Localization of the sites of synthesis and action of insulin-like growth factor-I in the rat uterus

In this report we have used in situ hybridization to localize insulin-like growth factor-I (IGF-I) expression in the uterus and have examined the effects of exogenous IGF on 3H-methyl thymidine incorporation into DNA, in uterine sections in organ culture. IGF-I mRNA was detected in all layers of the uterus but was particularly abundant in the outer longitudinal smooth muscle layer. Although IGF-I mRNA was detectable in untreated, immature rat uteri, the abundance in each layer of the uterus was increased after 17 beta-estradiol (E2) administration. A similar increase was seen in uteri from ovariectomized, hypophysectomized rats treated with E2. IGF-I when added to uterine sections in organ culture had no significant effect on DNA synthesis in the absence of E2. However, a dose-dependent increase in DNA synthesis was seen in the presence of E2. The half-maximal and maximal responses required 1 and 10 ng IGF-I, respectively. Autoradiographic localization of 3H-methyl thymidine incorporation into DNA demonstrated that the majority of the DNA synthesis occurred in the stromal layer. These findings are consistent with the hypothesis that IGF-I may function as a paracrine growth factor, mediating stromal-myometrial cell interaction.     

Down regulation of c-myc, c-fos and erb-B during estrogen induced proliferation of the chick oviduct

Oncogenes c-myc, H-ras, c-fos and erb-B were constitutively expressed in immature chick oviduct withdrawn from estrogen administration for 2.5 weeks after 10 d of primary estrogen stimulation. Following secondary estrogen stimulation of the withdrawn chicks, synthesis of egg white proteins is rapidly induced and remaining non-functioning tubular gland cells are stimulated to proliferate with a doubling time of 24 h. During first 12 h of secondary estrogen stimulation, H-ras mRNA levels doubled and did not increase further at 24 h and 48 h. The steady state levels of c-myc, erb-B and c-fos mRNA decreased 24 h following secondary estrogen stimulation. The levels of these oncogene RNAs in oviduct were similar at 48 h following secondary estrogen stimulation to those from immature chicks administered 10 d of primary estrogen stimulation. Thus elevated expression of c-myc and c-fos mRNA does not appear to be necessary components for sustained estrogen induced cell proliferation in the chick oviduct.     

Effect of estradiol on estrogen receptor expression in rat uterine cell types

In rodent uterus, both up- and down-regulation of estrogen receptor alpha (ERalpha) messenger ribonucleic acid (mRNA) and protein levels by estradiol has been demonstrated; however, it is not known which of the uterine compartments (endometrial epithelium, stroma, myometrium) respond to estradiol with autoregulation of ERalpha. The purpose of the present study was to investigate and compare the kinetics and cell type-specific effects of estradiol on uterine ERalpha expression in immature and adult rats. Ovariectomized female rats were injected s.c. with sesame oil or estradiol-17beta. Uteri were collected and analyzed for changes in ERalpha mRNA using RNase protection assays (RPA) and in situ hybridization using radiolabeled probes specific for ERalpha. Immunohistochemical analysis was performed with a polyclonal antibody specific to ERalpha. Expression of ERalpha in the uterine epithelial cells decreased at 3 and 6 h after estradiol administration to immature and adult rats, respectively. At 24 h, ERalpha mRNA levels in the immature and mature rat uterus were higher than pretreatment levels but returned to baseline by 72 h. Pretreatment with cycloheximide did not block the 3-h repressive effect of estradiol, suggesting that the estradiol-induced decrease in ERalpha mRNA occurs independent of new protein synthesis. A decrease in ERalpha mRNA and protein was also observed in uterine epithelia at 3 and 6 h after an estradiol injection to immature and adult rats, and intensity of both the in situ hybridization signal and the immunostaining in the epithelium increased at 24 and 72 h. However, the periluminal stromal cells in the adult uterus and the majority of stromal cells of the immature uterus appeared to have increased ERalpha expression. The results indicate that down-regulation of ERalpha in the epithelia and up-regulation of stromal ERalpha play a role in early events associated with estradiol-induced cell proliferation of the uterine epithelia.     

The effects of sequential administration of 17 beta-estradiol on the synthesis and secretion of specific proteins in the immature rat uterus

We report here results of a study of the effect of sequential administration of 1 microgram 17 beta-estradiol in vivo on the incorporation of L-[35S]methionine into specific proteins in vitro in the immature rat uterus. One-dimensional SDS-PAGE analysis of labeled secreted uterine proteins and cellular proteins extracted from the luminal epithelial and from the stroma plus myometrial uterine fractions revealed that estradiol preferentially stimulated the synthesis of 110 K, 74 K and 66 K secreted proteins, 180 K and 110 K epithelial proteins and a 175 K stroma-myometrial protein among others, while it decreased the relative rate of synthesis of a 32.5 K secreted protein and a 70 K stroma-myometrial protein. The 110 K protein, a secreted luminal epithelial protein whose labeling in vitro dramatically increased greater than 60-fold per mg endometrial DNA after in vivo estrogen stimulation, may be a useful marker for studying estrogen action in the luminal epithelium of the immature rat uterus. Comparison of the secreted proteins labeled at 28 h (4 h after a second injection) and at 54 h (6 h after a third injection) revealed that estradiol effected a sequential change in the pattern of synthesis of secreted uterine proteins in vitro. Comparison of the number and magnitude of changes in the synthesis of specific proteins in the luminal epithelium and the stroma plus myometrium revealed that protein synthesis in the luminal epithelium is clearly more responsive to estradiol and readily distinguishable from the responsiveness of the stroma plus myometrium.     

The kinetics of the incorporation of newly synthesized ribonucleic acid and protein into the ribosomes of the uterus of the oestrogen-stimulated immature rat.

The kinetics of the synthesis of the components of polyribosomes was investigated in the uterus of the immature rat responding to the administration of oestradiol-17 beta. The hormone brings about a rapid stimulation of the association of newly synthesized mRNA with uterine ribosomes, which is maximal 2-4 h after oestradiol administration and causes the aggregation of pre-existing ribosomes into polyribosomes. Despite the striking stimulation of rRNA synthesis 2-4 h after hormone treatment [Knowler & Smellie (1971) Biochem. J. 125, 605-614], the accumulation of new rRNA into ribosomes does not reach a peak until 12 h after administration. At this time, the incorporation of new ribosomal protein is also maximal. A second peak of incorporation of newly synthesized mRNA into polyribosomes follows the peak of ribosome synthesis and coincides with the oestrogen-activated synthesis of DNA.     

Effect of estradiol on the early incorporation of (3H) thymidine into uterine DNA on immature mouse.

The incorporation of [3H]thymidine into uterine DNA was markedly depressed within 10 to 30 minutes after intraperitoneal administration of 17beta-estradiol to immature mouse. Maximum inhibition occurred about 6 hours after the hormone was administered. Uterine DNA content and the amount of [3H]thymidine incorporated into the acid-soluble fraction was not affected during the period of hormone-induced inhibition. Moreover, the in vitro incorporation of [3H]thymidine by isolated estradiol-treated mouse uterus was blocked. In contrast to the uterus, 17beta-estradiol did not influence the incorporation of thymidine into mouse liver DNA. Evidence is presented to show that the incorporation of thymidine into uterine DNA was blocked initially by 17beta-estradiol.     

Diversity and complexity of uterine mRNA from rats of differing hormonal states.

The diversity and complexity of the uterine mRNA population has been compared at two different stages of uterine growth and development in the rat. Analysis by cDNA hybridization to homologous mRNA indicates that there are 8000 different sequences expressed in the immature rat uterus responding to just 4 h of oestradiol-17 beta-induced growth while the fully developed uterus expresses 36 000 sequences. As a check on our results, the complexity is re-analysed by hybridization of mercurated mRNA to total unique DNA. Analysis by this method indicates that the hormone-stimulated immature rat uterus contains 12 000 poly(A)-containing mRNA sequences while the fully differentiated adult tissue contains 53 000 diverse sequences.     

Regulation of proto-oncogenes in rat parotid acinar cells in vitro after stimulation of beta-adrenergic receptors

Stimulation of beta-adrenoreceptors in rat parotid acinar cells in vitro by the beta-adrenergic agonist isoproterenol induces steady-state levels of c-fos mRNA and c-fos protein in these cells. A dramatic increase in the steady-state levels of c-fos mRNA was observed at 60 min, followed by a decrease at 2 h with a second peak at 4 h. c-fos induction in rat parotid acinar cells in vitro seems to be mediated by cAMP. Increased levels of p53 and c-myc mRNA were detected only at 60 min. c-abl and c-sis were also induced by isoproterenol but in a pattern different from that seen with c-fos. c-abl was the only oncogene in rat parotid gland which showed increased expression after chronic isoproterenol treatment of rats. In rat parotid acinar cells we observed no correlation between DNA synthesis and c-fos induction.     

Estrogen regulation of creatine kinase-B in the rat uterus

Creatine kinase-B (CKB) synthesis is rapidly and specifically induced by estrogen in the uterus of the immature rat. This study indicates that this elevation is due at least in part to increases in the levels of mRNA for CKB. The stimulation of CKB mRNA levels is rapid (a 7- to 10-fold increase is detected 1-3 h after estrogen administration), but transient, as levels return to near control values by 6 h. Analysis of cDNAs to both uterine and brain CKB mRNA indicate that the same sequence is expressed in both tissues despite earlier observations of heterogeneity of the protein isolated from the two tissues. A 1.7-kilobasepair DNA fragment containing the CKB promoter and 5' flanking sequences confers estrogen sensitivity on expression of the bacterial chloramphenicol acetyl transferase gene in HeLa cells on cotransfection with an estrogen-receptor expression vector. However, the CKB promoter sequences lack any motif with convincing similarity to the currently accepted consensus estrogen response element GGTCAnnnTGACC.     

Regulation of proto-oncogene expression and deoxyribonucleic acid synthesis in granulosa cells of perifused immature rat ovaries.

The present series of experiments examined the effects of follicle-stimulating hormone (FSH) and insulin (IN) on granulosa cell (GC) proto-oncogene expression and DNA synthesis. In the first study, GCs were harvested from immature rat ovaries after 15, 30, or 60 min of perifusion and DNA synthesis (3H-thymidine incorporation) and proto-oncogene mRNA levels were determined. The presence of c-myc and c-fos proteins was localized within GCs immunocytochemically. GCs of control ovaries exhibited modest levels of DNA synthesis and proto-oncogene expression. FSH/IN not only stimulated DNA synthesis but also increased c-myc, c-fos, and c-jun mRNA levels and the percentage of cells staining for c-fos and c-myc proteins. The protein kinase inhibitor, 2-aminopurine (2-AP), inhibited the FSH/IN-induced increases in c-myc and c-fos mRNA levels, the percentage of cells staining for Myc and Fos protein, and DNA and protein synthesis. The effects of 48 h of perifusion with FSH in the presence or absence of IN were also examined. These treatments were selected because after 48 h of continuous exposure to FSH alone, estradiol-17 beta (E2) secretion is enhanced and 3H-thymidine incorporation is inhibited. Conversely, FSH/IN maintains 3H-thymidine incorporation for up to 48 h of perifusion culture without stimulating E2 (Peluso et al., Endocrinology 1991; 128:191-196). After 48 h of perifusion, both FSH and FSH/IN stimulated c-fos mRNA and protein levels. However, high levels of c-jun mRNA and protein were detected only within GCs of FSH/IN-treated ovaries.(ABSTRACT TRUNCATED AT 250 WORDS)