Regulation of nuclear binding of the avian oviduct progesterone receptor. Changes during estrogen-induced oviduct development, withdrawal, and secondary stimulation

The progesterone receptors from various stages of estrogen induced oviduct development, estrogen withdrawal, and secondary stimulation with estrogen were examined. The progesterone receptors were characterized for their biological function (i.e. capacity for nuclear translocation, nuclear binding, and effects on RNA polymerase II activity) as well as certain physical properties. The progesterone receptors from the undeveloped or partially developed oviducts (0 to 8 days of estrogen treatment) displayed little or no nuclear translocation and binding in vivo or in vitro. Similarly, progesterone showed little or no effect in vivo on RNA polymerase II activity at the early stages of development. As development progressed from 8 to 12 days of estrogen treatment, the above parameters rapidly increased to maximal levels and plateaued through day 23 of estrogen treatment. A marked decrease in these parameters occurred within 1 day of estrogen withdrawal. The reverse series of events occurred during secondary estrogen stimulation of 10-day-old withdrawn chicks. While the receptor concentrations increased rapidly to maximum values by 2 days of restimulation, receptor function did not return until day 4. Similarly, the effects of progesterone on RNA polymerase II activity reached maximal values by day 4. The progesterone receptor isolated from oviducts during development, estrogen withdrawal, and restimulation, displayed similar patterns of cell-free binding to chromatin and nucleoacidic protein as that observed in vivo supporting the nativeness of the in vitro binding assay. In contrast, the cell-free binding of these same progesterone receptor to pure DNA were not similar to the in vivo binding, i.e. no patterns (differences) in progesterone receptor binding were observed. These data support that protein DNA complexes and not pure DNA represent the native acceptor sites for oviduct progesterone receptor. Comparison of the progesterone receptor between the functional and nonfunctional states revealed no differences in the steroid affinity for the receptor, in the apparent pI of the species, or in the sedimentation of the receptor under high salt conditions. However, the nonfunctional receptors consistently displayed a deficiency in one of the two monomer molecular species (the B species) as determined by isoelectric focusing. These results suggest that both monomer species of progesterone receptor are required for biological activity. Interestingly, the 7S "aggregate" species of the progesterone receptor was constantly detected even when only one of the monomer species was present.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

In situ hybridization of ovalbumin mRNA in the chick oviduct reveals target cell specificity for estrogen and progesterone

An in situ hybridization method using paraffin-embedded sections was used to characterize the chicken oviduct cells synthesizing ovalbumin mRNA due to the action of estrogen and progesterne. The cytodifferentiation of the oviduct cells was induced by 17β-estradiol administration to newly hatched female chicks. To avoid possible effect of estrogen on the action of progesterone the chicks were withdrawn from the estrogen by six days withdrawal period without hormone treatment. Ovalbumin mRNA was not synthesized after a period of estrogen withdrawal. Administration of estrogen induced ovalbumin mRNA in the tubular gland cells. Administration of progesterone induced the expression of ovalbumin mRNA in the surface epithelial cells. It was also found that progesterone induced mucus producing goblet cells in the surface epithelium. Estrogen did not have an effect on the mucus production, which suggests that progesterone could induce the terminal differentiation of the goblet cells. We conclude that the expression of ovalbumin in the surface epithelial cells and in the tubular gland cells is specific for progesterone and estrogen, respectively.     

Estrogen Receptor Is Not Primarily Responsible for Altered Responsiveness of Ovalbumin mRNA Induction in the Oviduct From Genetically Selected High- and Low-Albumen Chicken Lines

The role of estrogen receptor on ovalbumin mRNA induction by steroid hormones was investigated in primary cultures of oviduct cells from estrogen-stimulated immature chicks of genetically selected high- and low-albumen egg laying lines (H- and L-lines). In experiment 1,the extent of ovalbumin mRNA induction and changes in estrogen and progesterone receptors were compared between the oviduct cells from H- and L-lines with or without steroid hormones in the culture medium. In experiment 2, the effect of estrogen receptor gene transfection on the induction of ovalbumin mRNA was studied in the oviduct cells from the L-line chicks. The results showed a close correlation of the changes in ovalbumin mRNA with the numbers of nuclear and total estrogen receptors in the oviduct cells but not with the numbers of nuclear and total progesterone receptors. Estrogen receptor gene transfection induced ovalbumin mRNA to a moderate extent in the absence of the steroid hormones. To our surprise, however, estrogen receptor gene transfection apparently suppressed the ovalbumin mRNA responsiveness to estrogen to a considerable extent. It was concluded, therefore, that the extent of estrogen receptor expression might not be primarily responsible for the differences in responsiveness to steroid hormones of oviduct cells from genetically selected H- and L-line chickens.     

INTERACTION OF ESTROGEN AND PROGESTERONE IN CHICK OVIDUCT DEVELOPMENT : I. Antagonistic Effect of Progesterone on Estrogen-Induced Proliferation and Differentiation of Tubular Gland Cells

Daily administration of estrogen to immature female chicks results in marked oviduct growth and appearance of characteristic tubular gland cells which contain lysozyme. Although a rapid increase in total DNA and RNA content begins within 24 hr, cell specific protein, lysozyme, is first detectable after 3 days of estrogen. Progesterone administered concomitantly with estrogen antagonizes the estrogen-induced tissue growth as well as appearance of tubular gland cells and their specific products, lysozyme and ovalbumin. When the initiation of progesterone administration is delayed for progressively longer periods (days) during estrogen treatment, proportionally greater growth occurs with more lysozyme and tubular gland cells after 5 days of total treatment. Progesterone does not inhibit the estrogen-stimulated increase in uptake of α-aminoisobutyric acid and water by oviduct occurring within 24 hr or the estrogen-induced increase in total lipid, phospholipid, and phosphoprotein content of serum. The above results of progesterone antagonism can best be explained by the hypothesis that progesterone inhibits the initial proliferation of cells which become tubular gland cells but does not antagonize the subsequent cytodifferentiation leading to the synthesis of lysozyme and ovalbumin once such cell proliferation has occurred.     

Hormonal regulation of a chicken oviduct messenger ribonucleic acid that shares a common domain with gizzard myosin light chain kinase

Changes in myosin light chain kinase (MLCK) and calmodulin (CaM) mRNAs have been evaluated during estrogen-mediated differentiation of the chicken oviduct. Also examined were acute changes that occur in oviduct RNA from animals stimulated with estrogen, withdrawn from hormone and then injected for 1, 2, and 4 days with synthetic estrogen [diethylstilbestrol (DES)], progesterone (P), or testosterone (T). Small changes were noted in both CaM and MLCK RNAs during primary stimulation when oviduct cells are actively dividing. On the other hand no significant changes were observed during secondary stimulation regardless of the steroid hormone injected. These data support the contention that CaM and MLCK are constitutively expressed but vary as a function of cell cycle. The MLCK mRNA is 5.5 kilobases (kb) but the MLCK cDNA also hybridizes to an oviduct RNA 2.7 kb long. This RNA species is acutely regulated by estrogen, P, and T but in a manner different from that of ovalbumin mRNA. The magnitude of stimulation of the 2.7 kb mRNA by diethylstilbestrol and T is greater than that of ovalbumin whereas changes in response to P are similar. The 12- to 16-fold increase of the 2.7 kb mRNA in response to T is the largest effect reported for this hormone acting on oviduct. The 2.7 kb mRNA encodes an unknown protein yet contains a 520 nucleotide segment that is highly homologous with the COOH-terminal coding portion of the MLCK mRNA. Since this homology does not include either catalytic or CaM-binding domains of MLCK, it is unlikely that the 2.7 kb mRNA encodes a CaM-dependent protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

The chick oviduct in tissue culture. I. Initial characterization of growing primary oviduct tissue cultures

A simple three-enzyme treatment of collagenase, dispase and hyaluronidase on finely minced chick oviduct yields clumps of 50-150 cells. These cells attach to collagen-treated dishes and survive in culture for at least 2 weeks without subculturing. Oviduct cell cultures can also be induced to grow. Estradiol or epidermal growth factor (EGF) induce a 40% increase in cells in 4 days when cultures are grown in serum levels that do not support growth. Serum from estrogen-stimulated chicks promotes rapid cellular proliferation (doubling times of 1-2 days). Sera from estrogen withdrawn chicks, laying hen or horse do not support as rapid proliferation. The oviduct growth-promoting factors in serum from estrogen-stimulated chicks are not steroids or fibroblast growth factors (FGF). Removal of steroids from these sera by charcoal treatment or delipidization does not decrease the rate of growth. The addition of 1-100 nM estradiol does not increase a serum's ability to promote growth. Purified FGF or platelet-derived growth factor (PDGF) do not induce oviduct proliferation. These results were reproduced in oviduct cell cultures started from estrogen-stimulated and withdrawn chicks as well as laying hens. Thus the factors in serum from estrogen-stimulated chicks that promote rapid oviduct growth are induced by estrogen treatments in vivo, but do not seem to be only steroids.     

Magnum morphogenesis during the natural development of the quail oviduct: analysis of egg white proteins and progesterone receptor concentration

The histological development of the quail oviduct and the changes in concentrations of progesterone receptor, ovalbumin, conalbumin, ovomucoid and ovoglycocomponents are analyzed during the period spanning 7-35 days of age. The initiation of luminal epithelial cell proliferation is the first event of magnum growth. The epithelial cells begin to evaginate into subepithelial stroma and form tubular glands. Meanwhile, luminal epithelium starts cellular pleomorphism through ciliogenesis. No egg white proteins are detectable in the developing glands; at the same time, the concentration of the progesterone receptor increases from about 5500 sites/cell to 30,300 sites/cell. Tubular gland cells then begin to synthetize and accumulate egg white proteins, mucous cells differentiate in the luminal epithelium, and the cell proliferation decreases and finally stops. Compared with earlier studies dealing with the blood levels of estrogen and progesterone in developing quails during the same period, and the cellular changes induced in the oviducts of ovariectomized and ovariectomized-hypophysectomized quail by exogenous steroids, these results distinguish between the cellular responses that are physiologically controlled by estradiol and other responses that have multihormonal regulation.     

Estrogen receptor levels in the oviducts and endometria of cynomolgus macaques during the menstrual cycle

We sampled oviducts and endometria of 27 cynomolgus macaques during the menstrual cycle and measured the concentration of nuclear and cytoplasmic estrogen receptors in these tissues by exchange assay. We assessed the stage of the cycle by correlating serum estradiol (E2) and progesterone (P), as measured by radioimmunoassay, with the morphological condition of the ovaries, oviducts and endometrium of each animal. We have previously identified a series of oviductal stages that reflected the orderly sequence of cytological changes in the oviduct during the cycle, and we normalized receptor measurements to these stages. The amounts of nuclear and cytoplasmic estrogen receptor in both the oviduct and the endometrium were approximately twofold greater in the follicular phase than in the luteal phase. In the follicular phase, elevated receptor levels were associated with oviductal proliferation and differentiation, as well as with endometrial proliferation. During the luteal phase, lowered levels were correlated with atrophy and dedifferentiation in the oviduct, but with hypertrophy and progestational development in the endometrium. When the luteal phase of one cycle ends and the follicular phase of the next begins, it is a decline in serum P, not a rise in serum E2, that precedes the elevation in estrogen receptor level and the onset of proliferation in the oviduct and endometrium. Proliferation of the reproductive tract and elevations in nuclear estrogen receptor levels during the early follicular phase can therefore be viewed as consequences of the release of the system from antagonism by P.     

Effects of serial hormone treatments on egg white protein synthesis. Further evidence of translational regulation

The administration of either progesterone or estrogen to withdrawn chicks several hours after a first dose of estrogen affected ovalbumin synthesis differently than its mRNA levels [S. S. Seaver (1981) J. steroid Biochem. 14, 949-957]. This suggested that the hormones were regulating the translation of ovalbumin directly. In this paper we report that serial hormone treatments also affect the rates of synthesis of two other egg white proteins, conalbumin and ovomucoid. When progesterone was administered 4 h after estrogen, conalbumin synthesis decreased. When either progesterone or a second dose of estrogen was administered 12 h after the first dose of estrogen, conalbumin synthesis increased. Serial hormone treatments did not always affect all three proteins similarly. At later times, administering progesterone after estrogen decreased ovomucoid synthesis but did not affect conalbumin or ovalbumin synthesis. To determine if the serial hormone treatments affect egg white protein mRNA's in a similar way, changes in ovalbumin and conalbumin mRNA levels were quantified in a rabbit reticulocyte cell-free translation system and were compared to changes in ovalbumin and conalbumin synthesis as measured in chick oviduct tissue minces. When serial hormone treatments were 12 h apart, ovalbumin and conalbumin synthesis was 50-300% higher than that predicted by the changes in ovalbumin or conalbumin mRNA levels. This is further evidence that translation of both conalbumin mRNA and ovalbumin mRNA is directly regulated by steroid hormones.     

Sexual maturation-associated and estrogen-induced progesterone receptor expression in the bursa of Fabricius

The expression of the progesterone receptor (PR) was studied in the chicken bursa of Fabricius (BF) in both sexes from the time of hatching until the bursal involution. Steroid binding studies, immunohistochemistry, and autoradiography were used to characterize and localize the receptor. Three different polyclonal antibodies (IgG-RB, IgG-G3, and IgG-RB2) directed against the chick oviduct progesterone receptor were used for the studies. With immunohistochemistry, no receptor-positive cells were detected in the bursae of young chicks. The first receptor-positive cells were occasionally seen at the age of 10 wk in the frozen sections, not in the paraffin sections. In older female chicks, the staining became more abundant. In males, the PR was expressed only after estradiol treatment. The staining was located in the nuclei of the subepithelial and the interfollicular cells, which were probably mesenchymal in origin. The bursal epithelium and the lymphocytes were not stained. By using a combined technique of autoradiography and immunohistochemistry, we were able to demonstrate that the same cells also concentrated tritiated ORG 2058 (a specific synthetic progestin) in their nuclei. In steroid binding studies with tritiated ORG 2058, the receptor concentration after the age of 10 wk was 50 to 120 fmol/mg protein. Low-level ORG 2058 binding was also detected in young chicks of both sexes before the age of 10 wk. The progestin-binding molecule resembled the progesterone receptor of the chick oviduct in molecular size (studied with HPLC) and binding properties. The PR expression in the BF was preceded by the expression of PR in the oviduct stromal cells and by an increase in oviduct epithelial proliferation, indicating the BF is affected by factors associated with sexual maturation. It is concluded that the subepithelial and the interfollicular stromal cells in the BF, but not the epithelial or follicular cells, are estradiol-sensitive in both sexes immediately after hatching. The endogenous estrogens, however, are not able to induce PR until after the onset of sexual maturation, and only in females. This implies that estrogen and progesterone may affect the structural organization of the BF through the stromal cells, but probably not before the onset of puberty.     

Retention of estrogen receptors in vitro requires limited estradiol exposure in vivo

Maintenance of functional estrogen receptors in culture has been accomplished in chick oviduct cells by manipulating the estrogen exposure before tissue dissociation. Tissue from chicks pre-treated with daily 17-beta-estradiol injections for 2 weeks or with 2 weekly diethylstilbestrol implants can be established in culture using a variety of enzymes. Tissue from animals with chronic estrogen stimulation must be withdrawn from hormone in culture at least 4 days before the digestion procedure. When tissue is digested using collagenase and pancreatin buffered by bovine serum albumin (Fraction V), large quantities of virtually fibroblast-free cultures can be established. The estrogen and progesterone receptors remain intact at normal levels using this procedure. The receptors have maintained biological function as evidenced by two hormone-dependent measurements. The first was an increase in the amount of ovalbumin mRNA transcribed in response to estrogen supplementation of the cultures compared to cultures with no estrogen. The second function was an increase in ovalbumin protein secreted into the medium upon estrogen stimulation. The protein increment demonstrated that the hormone-induced levels of mRNA were functional and capable of being translated.