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Oncogenes c-myc, H-ras, c-fos and erb-B were constitutively expressed in immature chick oviduct withdrawn from estrogen administration for 2.5 weeks after 10 d of primary estrogen stimulation. Following secondary estrogen stimulation of the withdrawn chicks, synthesis of egg white proteins is rapidly induced and remaining non-functioning tubular gland cells are stimulated to proliferate with a doubling time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.     

Estrogen-like effects of combined dexamethasone and tamoxifen in the chick oviduct

The effects of dexamethasone alone on withdrawn chick oviduct weight, DNA, protein content and progesterone receptor concentration were barely detectable, whereas ovalbumin and conalbumin synthesis were increased. When dexamethasone and tamoxifen were combined, a marked increase of total proteins, including egg white proteins, DNA and wet weight occurred. Progesterone receptor also was increased. The most striking result was the stimulation of DNA polymerase-α activity by combined dexamethasone and tamoxifen, whereas either compound was completely ineffective.     

The Relationship of the Estrogen Receptor to the Induction of Vitellogenin in Chicken and Xenopus Liver

The mechanisms involved in the regulation of gene expression in eukaryote cells, although an area of active research, are still largely unknown. This is at least partly due to the lack of good experimental model systems. One type of system which is being exploited with some considerable success is the induction of proteins by steroid hormones. Studies on the effects of estrogen and progesterone on the synthesis of the egg white proteins in the chick oviduct, for instance, have yielded substantial insight into both the regulation of protein synthesis by steroid hormones [1] and the arrangement of the DNA sequences coding for these proteins [2, 3].
The need for other good inducible systems clearly exists and the induction of vitellogenin, the precursor of the major egg yolk proteins, by estrogen in the livers of the chicken and frog ( Xenopus laevis ) is one that is attracting increasing interest. In common with the chick oviduct, large amounts of a specific protein are synthesised in response to a well defined hormonal stimulus. However, the induction of vitellogenin also has the advantage that the response is not complicated by the extensive hyperplasia that follows estrogen treatment in the chick oviduct [4, 5] and that vitellogenin may be induced in vitro [6–11].
The aims of this review are first to discuss recent data on the induction of vitellogenin and vitellogenin mRNA both in vivo and in vitro and then to relate this data to the properties of the estrogen receptor, present in chicken and Xenopus liver, which is thought to mediate the induction of vitellogenin by estrogen.     

Commitment of chick oviduct tubular gland cells to produce ovalbumin mRNA during hormonal withdrawal and restimulation

Acute withdrawal of estrogen from chicks leads to a precipitous decline in egg white protein synthesis and egg white mRNAs in the oviduct. In this paper we explore the biochemical basis of this phenomenon as well as the capacity of the "withdrawn" tubular gland cells to be restimulated with steroid hormones. During withdrawal, the decline in ovalbumin mRNA was closely correlated with the decline in nuclear estrogen receptors. Within 2-3 d of estrogen removal a withdrawn state was established and then maintained, as defined by a 1,000-fold-lower level of ovalbumin mRNA and a 20-fold-lower level of nuclear estrogen receptors, relative to the estrogen-stimulated state. The number of active forms I and II RNA polymerases declined by 50% during this time. Histological examination of oviduct sections and cell suspensions, combined with measurements of DNA content, revealed that tubular gland cells persisted as a constant proportion of the cell population for 3 d after estrogen removal. Despite a 1,000-fold decrease in the content of ovalbumin mRNA, the ovalbumin gene remained preferentially sensitive to digestion by DNase I. When 3-d-withdrawn oviducts were restimulated with either estrogen or progesterone, in situ hybridization revealed that greater than or equal to 98% of the tubular gland cells contained ovalbumin mRNA. Induction by a suboptimal concentration of estrogen was correlated with a lower concentration of ovalbumin mRNA in all cells rather than fewer responsive cells.     

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.     

Avian SERPINB11 gene: characteristics, tissue-specific expression, and regulation of expression by estrogen

Serpins, a group of proteins with similar structural and functional properties, were first identified based on their unique mechanism of action: their inhibition of proteases. While most serpins have inhibitory roles, certain serpins are not involved in canonical proteolytic cascades but perform diverse functions including storage of ovalbumin in egg white, transport of hormones (thyroxine- and cortisol-binding globulin), and suppression of tumors. Of these, serpin peptidase inhibitor, clade B, member 11 (SERPINB11) is not an inhibitor of known proteases in humans and mice, and its function is unknown. In the present study, the SERPINB11 gene was cloned, and its expression profile was analyzed in various tissues from chickens. The chicken SERPINB11 gene has an open reading frame of 1346 nucleotides that encode a protein of 388 amino acids that has moderate homology (38.8%-42.3%) to mammalian SERPINB11 proteins. Importantly, SERPINB11 mRNA is most abundant in the chicken oviduct, specifically luminal and glandular epithelia, but it was not detected in any other chicken tissues of either sex. We then determined effects of diethylstilbestrol (DES; a synthetic nonsteroidal estrogen) on SERPINB11 expression in the chicken oviduct. Treatment of young chicks with DES induced SERPINB11 mRNA and protein only in luminal and glandular epithelial cells of the oviduct. Collectively, these results indicate that the novel estrogen-induced SERPINB11 gene is expressed only in epithelial cells of the chicken oviduct and implicate SERPINB11 in regulation of oviduct development and differentiated functions.     

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.     

Establishment and characterization of Xenopus oviduct cells in primary culture

Based on a previously established procedure for Xenopus hepatocytes, we describe tubular oviduct cells in primary culture which continue to secrete substantial quantities of egg jelly for several days, as can be visualized microscopically. Freshly isolated cells exhibited a culture shock response [A. P. Wolffe, J. F. Glover, and J. R. Tata (1984) Exp. Cell Res. 154, 581], from which they recovered by the third day in culture. This recovery was characterized by (a) the diminished synthesis of heat shock proteins hsp 70 and hsp 85, (b) the cessation of the drop in number of estrogen receptor, and (c) the enhanced rate of synthesis of cellular and secreted proteins. The oviduct estrogen receptor had the same characteristics as those in other estrogen target tissues and was present in the same amount as in adult female Xenopus hepatocytes [A. J. Perlman, A. P. Wolffe, J. Champion, and J. R. Tata (1984) Mol. Cell. Endocrinol. 38, 51]. Unlike the latter in primary culture [M. P. R. Tenniswood, P. F. Searle, A. P. Wolffe, and J. R. Tata (1983) Mol. Cell. Endocrinol. 30, 329], oviduct cell cultures did not actively metabolize either estradiol or progesterone (t1/2 approximately equal to 55 and 7 h, respectively). The successful establishment and characterization of primary cultures of both liver and oviduct cells now fulfil the conditions required for investigating the basis for tissue specificity of regulation by estrogen of Xenopus egg protein gene expression in primary cell culture.     

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.     

Coordinated assembly of the developing egg

The developing vertebrate oocyte autonomously makes most of the components of the machinery for DNA and protein synthesis, as well as ‘maternal’ mRNAs, needed immediately after fertilization. However, specialized egg constituents such as yolk proteins, egg white, cholesterol, lipoproteins and egg coat substances, are formed in the liver, oviduct and perhaps in follicle cells. The assembly of the developing egg is therefore a fascinating example of division of labor among oocytes and extra-oocyte tissues, the coordination of activities of the latter being achieved by hormones.     

Oviduct-specific expression of tissue plasminogen activator in laying hens

Egg-laying hens are important candidate bioreactors for pharmaceutical protein production because of the amenability of their eggs for protein expression. In this study, we constructed an oviduct-specific vector containing tissue plasminogen activator (tPA) protein and green fluorescent protein (pL-2.8OVtPAGFP) and assessed its expression in vitro and in vivo. Oviduct epithelial and 3T3 cells were cultured and transfected with pL-2.8OVtPAGFP and pEGP-N1 (control vector), respectively. The pL-2.8OVtPAGFP vector was administered to laying hens via a wing vein and their eggs and tissues were examined for tPA expression. The oviduct-specific vector pL-2.8OVtPAGFP was expressed only in oviduct epithelial cells whereas pEGP-N1 was detected in oviduct epithelial and 3T3 cells. Western blotting detected a 89 kDa band corresponding to tPA in egg white and oviduct epithelial cells, thus confirming expression of the protein. The amount of tPAGFP in eggs ranged 9 to 41 ng/mL on the third day after vector injection. The tPA expressed in egg white and oviduct epithelial cells showed fibrinolytic activity, indicating that the protein was expressed in active form. GFP was observed only in oviducts, with no detection in heart, muscle, liver and intestine. This is the first study to report the expression of tPA in egg white and oviduct epithelial cells using an oviduct-specific vector.     

Effects of insulin-like growth factor-I,estrogen, glucocorticoid,and transferrin on the mRNA contents of ovalbumin and conalbumin in primary cultures of quail (Coturnix coturnix japonica) oviduct cells

The effects of estrogen, dexamethasone, insulin-like growth factor-I (IGF-I), and transferrin on the messenger RNA (mRNA) contents of ovalbumin and conalbumin in primary cultures of quail oviduct cells were investigated. In the absence of one of the above hormones or factors, a decrease in ovalbumin mRNA was prominent. In particular, removal of IGF-I and transferrin caused a significant effect. Studies using a combination of estrogen, dexamethasone, IGF-I and transferrin indicated that IGF-I cooperates with estrogen or dexamethasone and transferrin works with dexamethasone. Specifically, IGF-I enhanced ovalbumin synthesis or increased cellular ovalbumin mRNA content depending on its concentration in the medium in the presence of estrogen. However, the effects of estrogen, dexamethasone, IGF-I, and transferrin were not similarly observed with conalbumin mRNA. These results show that ovalbumin synthesis is controlled by estrogen or glucocorticoid with IGF-I or transferrin and that cellular ovalbumin mRNA content is also regulated by these hormones or transferrin. In contrast, conalbumin synthesis and cellular content of conalbumin mRNA are not affected by these hormones under the conditions of the present study.     

The induction of ovalbumin and conalbumin mRNA by estrogen and progesterone in chick oviduct explant cultures

Estrogen pretreated chick oviduct tissue can be restimulated in vitro by physiological concentrations of estrogen and progesterone. The rates of synthesis of the major egg white proteins, ovalbumin and conalbumin, as well as the cellular levels of their respective mRNAs, increase after characteristic lag periods; this confirms previously reported results in vivo and demonstrates that both the lag phenomena and the mRNA induction are a function of the direct interaction of steroids with oviduct cells.The antagonistic action of progesterone on an estrogen-mediated induction of conalbumin mRNA also occurs in vitro, and the kinetics of this response are examined. Progesterone terminates the estradiol-induced accumulation of conalbumin mRNA within 30 min after addition to the medium; progesterone alone or in combination with estrogen, however, is capable of inducing conalbumin mRNA after a 4 hr lag period. The temporary nature of this antagonism and the fact that it does not occur with ovalbumin induction indicate the complexity of the oviduct's response to steroids.The role of protein synthesis in the induction of both ovalbumin and conalbumin was examined by including protein synthesis inhibitors in the culture medium. Puromycin, cycloheximide, emetine, pactamycin and high salt all block the induction of both ovalbumin and conalbumin mRNA when added together with either estrogen or progesterone. The effect of puromycin is reversible. After the drug is removed from the medium, the mRNA accumulation begins with the same characteristic lag period seen when no inhibitors are added. When given 2 hr after estrogen, puromycin stops the accumulation of conalbumin mRNA within 30 min, whereas cycloheximide and emetine allow the mRNA to accumulate for another 2 hr before causing complete inhibition. There is no effect of protein synthesis inhibitors on the number of estrogen receptors localized in the nucleus. The data suggest a direct link between protein synthesis and the steroid-induced accumulation of specific mRNAs in this system.     

Egg white proteins.

1. Egg white proteins are the principal solutes present in egg white, making up approximately 10% of its weight. 2. They are globular proteins and most have acidic isoelectric points. 3. Many are glycoproteins with carbohydrate contents ranging from 2 to 58%. 4. Of the major egg white proteins, lysozyme is the only one having catalytic activity, but many have specific binding sites, e.g. for vitamins such as biotin, riboflavin and thiamin, or for metal ions such as FeIII. 5. A major group are those showing proteinase inhibitory activity, and they include ovomucoid, ovoinhibitor, cystatin and ovostatin. 6. The synthesis of egg white protein occurs in the oviduct, and is hormonally controlled either by oestrogens or progesterone. 7. Extensive studies have been carried out in the genes coding for egg white proteins.     

Purification of β-N-Acetylhexosaminidase from Egg White and the Microsomal and Lysosomal Fractions of Hen Oviduct

β-N-Acetylhexosaminidase (NAHase) was purified from egg white and the lysosomal and microsomal fractions of hen oviduct. The purification procedure included affinity chromatography using Sepharose 4B coupled with IgG specific for NAHase of hen oviduct. The isoelectric points of the three enzymes were different, but their antigen determinants were identical. In sodium dodecyl sulfate polyacrylamide gel electrophoresis, both the egg white and lysosomal enzyme gave only one protein band each, corresponding to a MW of 68000 and 53000, respectively, but the microsomal enzyme gave two protein bands, corresponding to those of the lysosomal and egg white enzymes.     

Exploring the chicken egg white proteome with combinatorial peptide ligand libraries

The use of two types of peptide ligand libraries (PLL), containing hexapeptides terminating either with a primary amine or modified with a terminal carboxyl group, allowed the discovery and identification of a large number of previously unreported egg white proteins. Whereas the most comprehensive list up to date ( Mann, K. , Proteomics 2007, 7, 3558- 3568 ) tabulated 78 unique gene products, our findings have almost doubled that value to 148 unique protein species. From the initial nontreated egg, it was possible to find 41 protein species; the difference (107 proteins) was generated as a result of the use of PLLs from which a similar number of species (112 and 109, respectively) was evidenced. Of those, 35 proteins were the specific catch of the amino-terminus PLL, while 33 were uniquely captured by the carboxy-terminus PLL. While a number of these low-abundance proteins might have a biological role in maintaining the integrity of the egg white and protecting the yolk, others might be derived from decaying epithelial cells lining the oviduct and/or represent remnants of products from the magnum and eggshell membrane components secreted by the isthmus, which might ultimately be incorporated, even if in trace amounts, into the egg white. The list of egg white components here reported is by far the most comprehensive at present and could serve as a starting point for isolation and functional characterization of proteins possibly having novel pharmaceutical and biomedical applications.     

Progesterone abbreviates the nuclear retention of estrogen receptor in the rat oviduct and counteracts estrogen action on egg transport

Progesterone has synergistic or antagonistic effects on several estrogenic actions. The effects of progesterone on estrogen-induced accelerated ovum transport and on the dynamics of estrogen receptors in the rat oviduct were examined. The involvement of the progesterone receptors in these phenomena was assessed. On Day 1 of pregnancy, rats were treated with estradiol, estradiol plus progesterone, or either one plus the progesterone receptor-blocking agent RU486. Control animals received the oil vehicle alone. The number of eggs remaining in the oviduct was assessed 24 h after treatment. Cytoplasmic and nuclear estrogen receptor levels in the oviduct, as well as plasma concentrations of estradiol and progesterone, were measured at various intervals--up to 11 h and 24 h after treatment, respectively. Accelerated oviductal egg transport induced by estrogen was blocked by the concomitant administration of progesterone. This effect of progesterone was not associated with changes in estrogen circulating levels and was preceded by a reduction in the total amount of estrogen receptors and by a shortened retention of estrogen receptors in the nucleus. The effects of progesterone on egg transport and on the levels of estrogen receptors were reversed by blocking the progesterone receptor with RU486, suggesting that both effects were receptor-mediated. These findings demonstrate that progesterone antagonizes the effect of estrogen on oviductal egg transport in the rat, and suggest that this antagonism is mediated by a reduction both in the amount of estrogen receptors and in their retention time in the nucleus.