Tubular Gland Cells Research Articles

Distribution of RNA Transcripts from Structural and Intervening Sequences of the Ovalbumin Gene

A study was made of the function of the intervening sequences in the ovalbumin gene, Radioactively labeled DNA probes for the intervening sequences were prepared and RNA's were isolated from whole cells, nuclei, and polysomes of estrogen-stimulated chick oviducts. The concentrations of messenger RNA (mRNA) transcripts from ovalbumin structural sequences (mRNAov) and transcripts corresponding to intervening sequences were then estimated by hybridization to cloned DNA probes. Oviduct tissue contains approximately 58,000 molecules of mRNAov sequences per tubular gland cell and most of these sequences are present in the cytoplasm. In contrast, there are 200 to 300 molecules of RNA per cell which are transcribed from the intervening sequences of the natural ovalbumin gene and almost all of these are found in the nucleus. The difference in distribution of structural and intervening sequence transcripts suggests that, unlike mature mRNA, the intervening sequences are not preferentially transported to cytoplasmic polysomes.

Steroid Hormone Regulation of Specific Gene Expression

This chapter describes the steroid hormone regulation of specific gene expression. A large number of proteins have been reported to increase in concentration or activity after steroid hormone treatment. In the chick, the administration of estrogen induces growth and differentiation and the synthesis of a number of egg white proteins, the major one of which is ovalbumin. The weight of the oviduct increases several hundredfold, and there is a massive proliferation of tubular gland cells. The oviduct proteins regulated by estrogen include conalbumin, ovomucoid, and lysozyme. The synthesis of these proteins, however, is not strictly coordinated, and each is synthesized more or less independently in response to estrogen. The rapidity of the vitellogenin response and the absence of any detectable vitellogenin messenger RNA (mRNA) in the untreated liver in both the xenopus and the rooster suggest a primary effect at the transcriptional level. The anamnestic response, on the other hand, is an observation that could be explained by a persistent change in the conformation of the vitellogenin gene and a permanent change in the estrogen-receptor generation or response.

Regulation of Gene Expression in the Chick Oviduct by Steroid Hormones

One of the central problems in our understanding of steroid hormone action is the absence of a molecular connection between the nuclear localization of hormone receptors in target cells and the subsequent accumulation of specific mRNAs in those cells. This paper deals with two aspects of this problem. First, we will describe experiments that demonstrate a pronounced lag between the time steroid receptors reach the nucleus and the induction of specific mRNAs. The existence of a lag has important implications because it suggests that there are as yet undefined, rate-limiting nuclear events interspersed between receptor binding in the nucleus and the dramatic acceleration of mRNA production that occurs a few hours later. This lag phenomenon is prevalent in steroid-responsive systems (see Palmiter et al. 1976 for references). However, it is not universal. For example, within the tubular gland cells of the chick oviduct, conalbumin mRNA (mRNAcon) is induced by estrogen...

Relationship of nuclear estrogen receptor levels to induction of ovalbumin and conalbumin mRNA in chick oviduct.

Oviduct nuclei from laying hens, or from chicks given an optimal dose of estrogen, contain receptors with a high affinity for 17beta-estradiol (apparent Kd of about 3.7 nM) at a concentration of about 10,600 molecules per tubular gland cell. Chicks withdrawn from estrogen stimulation exhibit a 7-fold lower level of nuclear estrogen receptors. Radioimmunoassay of 17beta-estradiol in the serum indicates that birds withdrawn from estrogen stimulation have a serum level of 0.09 nM. A maximum concentration of oviduct nuclear receptors is achieved when the serum level reaches 0.7 nM 17beta-estradiol. Endogenous 17beta-estradiol in the serum of laying hens is also approximately 0.7 nM. The concentration of nuclear estrogen receptors achieved by administering different dosages of 17beta-estradiol, 17beta-estradiol-benzoate, or diethylstilbestrol is related to the rate of accumulation of ovalbumin and conalbumin mRNA. The rate of conalbumin mRNA production is directly proportional to the concentration of nuclear receptors, i.e. half-maximal induction is obtained with about 5,300 nuclear receptors per tubular gland cell. In contrast, half-maximal induction of ovalbumin mRNA occurs when nuclear receptor levels are 80% of maximum; this is achieved with a dose of estrogen about 2.5 times that required for half-maximal conalbumin mRNA induction. These differential responses may be related either to different numbers of specific binding sites regulating the production of each mRNA, or to different affinities of regulatory sites for estrogen receptors.

A significant lag in the induction of ovalbumin messenger RNA by steroid hormones: A receptor translocation hypothesis

Although ovalbumin and conalbumin mRNA accumulate in the same tubular gland cells of the chick oviduct in response to estrogen or progesterone treatment, the kinetics of induction are markedly different. Conalbumin mRNA begins to accumulate within 30 min after estrogen administration, whereas there is a lag of approximately 3 hr before ovalbumin mRNA begins to accumulate, as measured by three independent assays. The kinetics of estrogen-receptor binding to chromatin indicate that these sites are saturated within 15 min of estrogen administration to the chicks, demonstrating that the lag is not due to slow uptake of the steroid. Suboptimal doses of estrogen produce the same lag, but the resultant rate of ovalbumin mRNA accumulation is lower than with an optimal dose. Partial induction of ovalbumin mRNA by a low dose of estrogen does not shorten the lag with an optimal dose. With progesterone, there is a lag of about 2 hr before either ovalbumin or conalbumin mRNA beginsto accumulate. Treatment of chicks with hydroxyurea shortens the lag for ovalbumin induction with either hormone. Inhibition of protein synthesis with emetine does not prevent the accumulation of either ovalbumin or conalbumin mRNA. With cycloheximide, however, ovalbumin mRNA accumulation can be prevented. The existence of a lag suggests that there are intermediate steps between the binding of steroid receptors to chromatin and the induction of ovalbumin mRNA. There are basically two models to explain these delays in response: one involving the accumulation of an essential intermediate, and the other involving a rate-limiting translocation of steroid receptors from initial nonproductive chromatin-binding sites to productive sites. Several aspects of the kinetics of ovalbumin mRNA induction are more consistent with the latter model.

Modulation of the synthesis in vitro of a hormone-induced protein by transfer RNA

WE have shown that transfer RNAs (tRNAs) and the enzymes which modify their structure at the macromolecular level (tRNA methyltransferases) are under hormonal control in the target tissue1–3. Oestrogen-induced, cell-specific protein synthesis in immature chick oviducts has been extensively studied4–6. Oestrogen induces cytodifferentiation of the primitive oviduct and the consequent synthesis by the tubular gland cells of ovalbumin, conalbumin, ovomucoid and lysozyme7. Continuous presence of oestrogen is required for the sustained synthesis of these proteins in immature chicks; withdrawal of oestrogen is accompanied by a gradual decline in cell-specific protein synthesis6. The tubular gland cells in the oviduct magnum, however, continue synthesising non-secretory proteins. Readministration of oestrogen to chicks after withdrawal (secondary stimulation) results in the restoration of the synthesis of cell-specific proteins without concomitant need for DNA synthesis. We report here that in an in vitro tRNA-dependent protein-synthesising system from Ehrlich ascites cells, stimulation of the translation of ovalbumin mRNA was significantly enhanced by tRNA extracted from the oviduct of oestrogen-stimulated immature chicks or from the oviduct of laying hens compared with tRNA from oviducts of withdrawn chicks. Therefore, the efficiency of synthesis in vitro of a hormone-induced protein can be regulated by tRNA.

The effects of estradiol and progesterone on the growth and differentiation of the quail oviduct.

The effects of estradiol and progesterone treatment on the growth and differentiation of the quail oviduct have been investigated in terms of morphology, growth, and biochemical differentiation. Histologically, estradiol induced the formation of tubular gland cells by 5 days of treatment, and epithelial differentiation by 10 days of treatment. Progesterone treatment appeared to enhance epithelial differentiation slightly, compared with continued estrogen treatment. The estradiol treatment also enhanced the growth of the oviduct, as indicated by increases in size, weight, and the content of the tissue constituents protein, RNA, and DNA. Estradiol treatment also induced the synthesis of two specific proteins, ovalbumin and lysozyme; however, only progesterone induced the synthesis of the protein avidin. Withdrawal from both types of treatment for 10 days caused a loss of cells and of tissue constituents. This loss was greater in those birds withdrawn from sequential estrogen-plus-progesterone treatment than in those withdrawn from estrogen treatment. In some cases, specific proteins were also absent in oviducts from birds withdrawn from treatment.

Induction of ovalbumin and conalbumin synthesis in immature chick oviducts by ethionine.

PROLONGED feeding of rats with ethionine results in a high frequency of hepatic carcinoma. Ethionine induces other pathological manifestations in other organs and tissues of experimental animals1, and some of its effects can be reversed rapidly by the administration of methionine. At the molecular level ethionine induces a rapid decrease in hepatic ATP concentration2–4, followed by inhibition of RNA5 and protein synthesis6. The decrease in ATP concentration is due to formation of S-adenosyl-ethionine7, an inhibitor of tRNA methyltransferases8,9, and so tRNA isolated from the liver of rats injected with adenine and ethionine is hypomethylated10,11. It is well established that several eukaryotic mRNAs are methylated on the 5′ terminus and that such modification is essential for translation12,13. To study the mechanism of methylation we attempted to produce a specific methyl-deficient mRNA by the administration of ethionine. To monitor the effectiveness of the deprivation of methyl groups the synthesis of hormone-induced ovalbumin in immature chick oviducts was followed. Administration of oestrogen to immature chicks causes cytodifferentiation and growth of the primitive oviduct14–17. The tubular gland cells synthesise ovalbumin, conalbumin, ovomucoid and lysozyme which comprise 85–90% of the egg-white proteins18. The continuous presence of oestrogen is required for sustained synthesis of these proteins in immature chicks; withdrawal of oestrogen is accompanied by a gradual decline in cell-specific protein synthesis as well as in the weight of the oviduct and the RNA content of the tissue17. But, the tubular gland cells in the oviduct magnum during withdrawal are retained although they do not synthesise cell-specific secretory proteins17,19. Readministration of hormone to chicks after withdrawal (secondary stimulation) results in restoration of cell-specific protein synthesis without concomitant need for DNA synthesis17. During these studies ethionine unexpectedly simulated the effects of the injection of oestrogen for secondary stimulation.

Induction of ovalbumin mRNA sequences by estrogen and progesterone in chick oviduct as measured by hybridization to complementary DNA.

A complementary DNA synthesized from ovalbumin mRNA was used in hybridization experiments to study the early effect of estrogen and progesterone on the accumulation of ovalbumin mRNA sequences in the chick oviduct. Chicks treated with estrogen withdrawn from the hormone maintain a steady level of 60 molecules of ovalbumin mRNA per tubular gland cell, at least 80% of which are localized in the cytoplasm. After estrogen administration, there is a 3- to 4-hour lag before a rapid increase in the number of ovalbumin mRNA sequences and a parallel increase in ovalbumin synthesis. Progesterone causes a more rapid increase in both ovalbumin mRNA sequences and ovalbumin synthesis with a lag period of only 90 min. The hybridization results demonstrate that both estrogen and pregesterone affect the amount of ovalbumin mRNA per cell. The 3-hour lag period seen with estrogen appears to be caused by some event after the binding of the estrogen receptor to chromatin but prior to change in the rate of transcription of the ovalbumin gene.

Interactions du benzoate d'oestradiol et de la progesterone sur le developpement de l'oviducte de caille ( Coturnix coturnix japonica) : II. Etude ultrastructurale des deux types de responses obtenues selon la dose de benzoate d'oestradiol injectée

The effects of various doses of estradiol benzoate on the magnum of immature and castrated quails have been studied by electron microscopy. With doses below 20 μg/day, which do not disturb the lipids and steroids metabolism, estradiol benzoate induices cellular multiplication, gland formation, and ciliogenesis in epithelial cells. Cytodifferentiation of tubular gland cells occurs (number of ribosomes increases and there is a development of endoplasmic reticulum and Golgi apparatus) but these cells do not produce secretions even after 12 days of treatment. Higher doses, equal or superior to 20 μg/day not only induce mitosis and gland formation, but also, a stimulation of the secretory processes in tubular glands cells and epithelial cells in which mucous granules appear. These secretory processes increase according the dose. Two types of in vivo responses to estrogen can be observed, depending on the injected dose.

Interactions du benzoate d'oestradiol et de la progesterone sur le development de l'oviducte de caille ( Coturnix coturnix japonica) : I. Etude pondèrale et histologique

The effects of estradiol benzoate (O.) and progesterone (P.) alone and in combination was investigated in quail ( Coturnix coturnix japonica) oviduct growth. At physiological doses, O. induced the differentiation of only two distinct cell types of the undifferentiated epithelium: ciliated cells and tubular gland cells. When injected with O., P. inhibited epithelial cell multiplication, cilia formation and tubular gland differentiation. But P. induced the differentiation of the goblet cells and played the most important part in the formation of secretory granules. Thus, after the activation of undifferentiated epithelial cells by estrogen, O. and P. were necessary at every stage to arrive at a functional unit.

Use of a specific probe for ovalbumin messenger RNA to quantitate estrogen-induced gene transcripts.

DNA complementary to purified ovalbumin messenger RNA (cDNA ov) was synthesized in vitro using RNA-directed DNA olymerase from avian myeloblastosis virus. This cDNAov was then employed in hybridization assays to determine the effect of estrogen on the number of ovalbumin mRNA (MRNAov) molecules per tubular gland cell of the chick oviduct. The changes in mRNAov were measured in immature chicks during primary stimulation, after hormone withdrawal and again following secondary stimulation of the chick oviduct with estrogen. The number of mRNAov per tubular gland cell was also determined for egg-laving hen. Daily estrogen administration to the immature chick resulted in growth of the oviduct, differentiation of epithelial cells to tubular glands, and a corresponding increase in the concentration of mRNAov in the tubular gland cell from essentially zero before estrogen administration to 48,000 molecules per cell after 18 days of estrogen treatment. Upon withdrawal of estrogen from the chick, the mRNAov concentration decreased to a level of 0-10 molecules/tubular gland cell after 12 days. Readministration of a single dose of estrogen to these chicks resulted in a dramatic and rapid increase in the concentration of mRNAov. Within 30 min, the mRNAov concentration approximately doubled and by 29 hr the tubular gland cell concentration had reached 17,000 molecules. The initial transcription rate for the ovalbumin gene was 12 mRNAov molecules/min. With these data, we have calculated that the half-life of the ovalbumin messinger RNA should be on the order of 40-60 hr and that the steady-state concentration of mRNAov per tubular gland cell was 50,000 molecules. Similarly, each messenger RNA molecule was translated approximately 50,000 times during its lifetime in order to effect the necessary quantity of ovalbumin required for egg production. These data substantiate the hypothisis that estrogen exerts its primary action at the level of transcription to effect the synthesis of nascene mRNA molecules which in turn code for synthesis of hormone-induced proteins.

Steroid hormone regulation of specific messenger RNA and protein synthesis in eucaryotic cells.

Evidence is presented that the induction of specific proteins in the chick oviduct by the steroid hormones estrogen and progesterone, involves a primary effect at the level of gene transcription. The intracellular levels of mRNA's which code for the synthesis of the egg-white proteins, ovalbumin and avidin, have been quantitated in a heterologous protein synthesizing system. It is demonstrated that these levels are directly dependent upon the inducing steroid, estrogen or progesterone, respectively. Ovalbumin mRNA has been purified to apparent homogeneity. This ovalbumin mRNA was then used as a template for the synthesis of a complementary DNA copy catalyzed by the enzyme reverse transcriptase which was isolated from avian myeloblastosis virus. This radioactively labeled complementary DNA was used to demonstrate, by means of DNA excess hybridization, that the ovalbumin gene is represented only once in each haploid genome of the chick cell. Next the complementary DNA copy of the ovalbumin mRNA was used as a genetic probe to determine the precise number of sequences of ovalbumin mRNA present at any one time after the administration of estrogen. It was demonstrated that the unstimulated chick contained no sequences of ovalbumin mRNA. Within a very short period of time after estrogen is administered the ovalbumin sequences begin to appear and reach a steady state level of 140,000 molecules per tubular gland cell. It could also be calculated that each ovalbumin molecule is probably translated some 50,000 times during its life which explains why ovalbumin comprises some 60% of the total protein in the oviduct cell. Following withdrawal of the oviduct from estrogen treatment, ovalbumin mRNA sequences again drop to undetectable levels. However, following a single injection of estrogen to these withdrawn animals, new ovalbumin mRNA sequences could be detected within 30 minutes. These data suggest that estrogen controls the activity of the ovalbumin gene via a pure transcriptional control mechanism. It is also demonstrated that the efficiency of the complementary DNA as a means of quantitating specific mRNA sequences is some 1,000 times more sensitive than the best available in vitro translation system. Finally, the efficacy of four popular translation systems is compared. It is suggested that for initial studies involving hormonal control of mRNA levels, the translation system derived from wheat germ is the simplest and most sensitive.

Fluorescent antibody localization of the vitamin D-dependent calcium-binding protein in the oviduct of the laying hen.

Fluorescein isothiocyanate-conjugated sheep antirabbit gamma-globulin was used in the indirect fluorescent antibody technique to localize the vitamin D-dependent calcium-binding protein (CaBP) in the shell gland of the laying hen. Intense specific fluorescence, indicative of the presence of CaBP, was found only in the tubular gland cells and not in the surface epithelium. CaBP was primarily associated with the luminal surface and other cell boundaries, and in lesser concentrations within the cells except for occasional intracellular regions of high concentration. The magnum, isthmus and vagina were essentially negative. Since the concentrations of intestinal CaBP is highly and directly correlated with the efficiency of calcium absorption, the present results suggest that the tubular gland cells might have a primary role in the transport of calcium associated with egg shell formation.

Ovalbumin Messenger RNA: Evidence That the Initial Product of Transcription Is the Same Size as Polysomal Ovalbumin Messenger

The messenger RNA for ovalbumin, the major secretory protein of the chick oviduct, appears not to be made as a high-molecular-weight precursor when artifacts due to aggregation are eliminated. No ovalbumin messenger RNA sequences that will hybridize to complementary DNA made against ovalbumin mRNA are found in concentrated samples of hen oviduct RNA larger than 28 S. The sensitivity of the hybridization assay is sufficient to detect less than one molecule of ovalbumin mRNA precursor per tubular gland cell. Newly synthesized ovalbumin messenger RNA isolated from immature chicks stimulated briefly by estrogen is the same size as that found in hen polyribosomes. We conclude that ovalbumin messenger RNA does not undergo any significant change in molecular weight from its initial transcription to its incorporation into polyribosomes.

Quantitation of ovalbumin mRNA in hen and chick oviduct by hybridization to complementary DNA. Accumulation of specific mRNA in response to estradiol.

DNA complementary to ovalbumin mRNA was synthesized using reverse transcriptase from avian myelobastosis virus and ovalbumin mRNA, purified 60–70‐fold with respect to polysomal RNA, as template. The quantitative distribution of ovalbumin mRNA sequences in hen and chick oviduct was determined by hybridization with ovalbumin complementary DNA (cDNA). In hen, we calculate that there are approximately 36000 ovalbumin mRNA molecules per oviduct tubular gland cell. The polysome fraction contains 93% of ovalbumin mRNA; nuclear and non‐polysomal cytoplasmic RNA fractions contain 5% and 2% respectively. In the nucleus, the majority of sequences exist as the 16‐S “translatable” form when analyzed on formamide gradients. High‐molecular‐weight RNA species (> 30 S) were detected, and contained less than 2% of total nuclear mRNA sequences. The appearance of ovalbumin mRNA sequences in total, nuclear and polysomal RNA fractions from chick oviduct was measured in response to estradiol. Prior to hormone treatment, low levels of sequences were present in all fractions, approximately one mRNA molecule per gland cell being detected in nuclei. Up to 2 h after treatment a small increase in ovalbumin mRNA concentration was observed in nuclear and, to a lesser extent, in polysomal RNA. Between 3 and 6 h ovalbumin mRNA accumulates rapidly in the polysome fraction and after 9 h the rate of appearance of this mRNA is approximately 7 molecules per min per gland cell. Our results indicate that the induction by estradiol of ovalbumin synthesis in oviduct is mediated either by the synthesis of new ovalbumin gene transcripts or the stabilization of ovalbumin gene products.

Histological changes in the regressing reproductive organs of sexually mature male and female Japanese quail.

A reduction of daily photoperiod from 16 to 6 per 24 h induced accelerated involution (regression) of the oviducts and ovaries in sexually mature Japanese quail. Sections taken from the oviduct shortly after cessation of egg laying, when examined histologically, indicated reduced cell size of the entire surface epithelium and cessation of secretory activity in the magnum and isthmus sections. Reduction in the size and apparent degeneration of the tubular gland cells was associated with an increased proliferation of the connective tissue in the magnum, isthmus, and uterus. Massive inclusions closely associated with the tubular gland cells characterized the regressing magnum, whereas large lumina filled with cellular debris and degenerating cells were seen in the isthmus and uterus. Regressing ovarian tissue was characterized by increased connective tissue and various forms of follicular atresia although primary oocytes and small follicles remained a constant feature. Signs of involution (regression) were seen in the majority of the testes, primarily, 10 days after light reduction. Seminiferous tubules filled with necrotic debris and increased steatogenesis were features associated with testicular involution (regression). Four weeks after light reduction, the majority of the lipoidal seminiferous tubules were cleared of necrotic debris and contained essentially a single layer of germinal epithelium. These results indicate that after 4 wk of decreased photoperiod the reproductive organs of both sexes were histologically similar to the organs during the immature stages of development.

Rate of Ovalbumin Messenger Ribonucleic Acid Synthesis in the Oviduct of Estrogen-primed Chicks

Abstract The concentration of translatable ovalbumin messenger RNA in the tubular gland cells of the chicken oviduct was determined from estimates of relative rate of ovalbumin synthesis, polysome concentration, size of ovalbumin-synthesizing polysomes, DNA concentration, and percentage of tubular gland cells in the oviduct. During secondary stimulation with estrogen, the concentration of ovalbumin messenger RNA rose from undetectable levels to approximately 70,000 molecules per tubular gland cell after 5 days when a steady state was reached. The initial rate of ovalbumin messenger RNA accumulation was about 22 molecules per min per tubular gland cell. Nearly all the translatable ovalbumin messenger RNA was localized in polysomes, even at very early times after hormonal stimulation, suggesting that there is no free pool of untranslated messenger. The half-life of ovalbumin messenger RNA was estimated to be about 24 hours by labeling the RNA in vivo followed by subsequent purification and determination of its specific activity. Two other potential approaches for determining messenger half-life were shown to be unsuitable. Using the half-life value of 24 hours, the steady state rate of ovalbumin messenger RNA synthesis was about 34 molecules per min per tubular gland cell. Based on theoretical values for the maximum rate of messenger RNA transcription in eukaryotes, one ovalbumin gene per haploid genome is sufficient to maintain the observed rate of ovalbumin messenger RNA synthesis.

Ovalbumin mRNA, complementary DNA and hormone regulation in chick oviduct.

ABSTRACT Chick oviduct differentiation is controlled by various steroid hormones, including both oestrogen and progesterone. Administration of oestrogen to chicks results in differentiation of tubular gland cells, which synthesize differentiated cell products, including ovalbumin and conalbumin. Ovalbumin can constitute as much as 60% of the protein synthesized by oviduct in the fully stimulated chick. Methods have been developed that allow for the quantitative assay of ovalbumin mRNA, the specific isolation of ovalbumin synthesizing polysomes by use of immunologic techniques, as well as the synthesis of a DNA probe complementary to the ovalbumin mRNA. With these techniques we have found that the rate of ovalbumin synthesis is proportional to the content of ovalbumin mRNA. Hybridization studies using the DNA probe indicate that there are only two copies of the ovalbumin gene per genome, and that there is no amplification of the ovalbumin genes to account for the large amount of ovalbumin synthesized.

Synergistic effects of oesterogen and progesterone on ovomucoid and conalbumin mRNA synthesis in chick oviduct.

OESTROGEN, progesterone and testosterone regulate the cytodifferentiation of the tubular gland cells which synthesise the majority of the egg white proteins and modulate specific protein synthesis1–6. Once tubular gland cells have differentiated in the presence of oestrogen, the synthesis of the egg white proteins can be induced by either oestrogen or progesterone3, 6. We reported that the synthesis of two of these proteins, conalbumin and ovomucoid, is maximally stimulated only with combinations of oestrogen and progesterone6. The evidence suggested that these effects were synergistic rather than simply additive. In order to answer the question of whether these synergistic effects were at the level of mRNA translation or mRNA synthesis, we isolated and quantified the messengers for these proteins by assaying them in a protein-synthesising system derived from rabbit reticulocytes7. Here we show directly that the different rates of specific protein synthesis reflect different mRNA concentrations.