Avidin Synthesis Research Articles

Chick oviduct differentiation. The effect of estrogen and progesterone on the expression of progesterone receptor

Progesterone receptor (PR) is a marker of estrogen action. Its cellular appearance during estrogen (20 mg/kg i.m.)-induced differentiation of the immature chick oviduct was therefore studied by immunohistochemistry. PR was located in the epithelial, mesothelial, submucosal stromal and smooth muscle cells. Progesterone (20 mg/kg i.m.) caused an obvious decrease in PR immunoreactivity without inducing synthesis of progesterone-dependent avidin. Thus mere receptor occupation by ligand is not sufficient for this induction. This paper reports that the expression of PR in the mucosal stromal cell differs from that in other cell types. In the mucosal stromal cell PR was inducible, i.e., not shown without the action of estrogen. The formation of tubular glands did not commence before mucosal stromal cells expressed PR. It would seem that the mucosal stromal cells have a crucial role in mediating epithelial differentiation. The onset of differentiation was preceded by vascularization and invasion of mononuclear cells in the submucosa. It was conspicuous that the smooth muscle cells of arteries also contained PR.

Distribution of estrogen and progesterone receptors and steroid-regulated gene products in the chick oviduct

The distribution of estrogen and progesterone receptors (ER and PR, respectively) was studied immunohistochemically in the chick oviduct. Estrogen receptor immunoreactivity was found only in the nuclei of glandular epithelial cells. Progesterone receptor was found in the nuclei of glandular and luminal epithelia, stroma, smooth muscle cells and in the mesothelium. The dissimilar distribution of ER and PR suggests that either ER concentration in the luminal epithelium and smooth muscle is very low (below the sensitivity of ER immunostaining) or that estrogens control their PR synthesis indirectly via ER in glandular cells. A known estrogen-inducible protein, ovalbumin, was localized in the same glandular epithelial cells as ER. A progestin-inducible protein, avidin, was found in part of the luminal and glandular epithelium cells but not in other PR-positive cell types. This indicates the importance of cellular differentiation in the regulation of avidin synthesis. Estrogen and progesterone administration had effects also on ER and PR immunoreactivity. Estrogen and progesterone administrations for 24 h decreased markedly the immunoreactivity of their receptors. The decrease in receptor immunoreactivity is most likely due to a transient loss of immunoreactive receptor protein, since the antibodies (H222, PR6) react both with transformed (4 S) and non-transformed (8 S) receptor forms. At the subcellular level, PR was localized in the chromatin by immunoelectron microscopy. Progestin administration seemed to decrease PR immunoreactivity especially in the heterochromatin area, suggesting that conformational chromatin rearrangements occur during down-regulation of PR.

Development of progestin-specific response in the chicken oviduct.

Avidin is a host acute defense protein induced by progestins and by inflammation caused by injurious factors such as microbes, viruses, toxic factors or tissue trauma. In the reproductive tract of egg-laying vertebrates avidin has evolved into a progestin-dependent secretory protein involved in anti-microbial action through its biotin avidity. For "progestin-dependent avidin" production, cellular differentiation by estrogen is necessary. In contrast, the expression of "progestin-independent or inflammation-induced avidin" does not require differentiation. Many cell types such as macrophages, heterophils and fibroblasts can produce avidin after non-specific cellular injuries. The wide distribution of avidin in avian, reptilian and amphibian species could be explained on the basis of its vital functions such as antimicrobial or antifungal, metabolic and immunomodulatory actions. The ontogeny of the progestin-dependent avidin synthesis is a complex event involving oviductal differentiation by steroid hormones leading to a specific gene expression. The first phase in oviductal differentiation by estrogens is characterized by a new chromatin organization and by an infiltration of progesterone receptor (PR)-containing mesenchymal cells into the subepithelial mucosa leading to epithelial cell differentiation ("mesenchymal and epithelial cell interaction"). The second phase in the differentiation of progestin-induced response is dependent on the presence of PR in the secretory cells. Two kinds of PR expression occur in the oviduct. The first is a "constitutive PR" and is found in the epithelial, submucosal and peritoneal cells of the immature chick oviduct without steroid treatment, and the second is an "inducible PR" found especially in the mucosal mesenchymal and smooth muscle cells. Avidin production requires PR in the target cells, but not all PR-containing cells can produce avidin. Therefore, in addition to PR, other transcription factors are needed to define the target cell specificity of the response to progestins. Earlier biochemical studies suggested that cytosolic and/or nuclear unoccupied PR was complexed as an 8 S form with the heat shock protein 90 (hsp90). Our immunohistochemical results, however, indicate that PR in vivo is not bound to hsp90, which is located entirely in the cytoplasm, whereas PR is an entirely nuclear protein in both ligand-occupied and unoccupied forms. Therefore, we assume that PR is a monomeric (4S) or homodimeric (5S) (chromatin?) protein associated to DNA. Ligand binding to PR appears to lead to a conformational change, dimer formation, tighter binding to PRE (progesterone responsive element) and to transcription factors, phosphorylation and proteolysis of PR as well as a chromatin change.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of avidin and other biotin-binding proteins in the deposition and distribution of biotin in chicken eggs. Discovery of a new biotin-binding protein.

In addition to the previously characterized egg-yolk biotin-binding protein (BBP-I), we have discovered another BBP (BBP-II) in the plasma and yolk from laying hens. BBP-I is stable to 65 degrees C, whereas BBP-II is stable to 45 degrees C. Both proteins are normally saturated with biotin and together they account for most, if not all, of the biotin in hen plasma and yolk, except in hens fed excessive amounts of biotin (greater than 1 mg of biotin/kg of feed). The maximal production of BBP-I is attained at lower levels of dietary biotin (approximately 50 micrograms/kg) than for BBP-II (approximately 250 micrograms/kg); however, the maximal production of BBP-II is severalfold greater than for BBP-I. Consequently, as dietary biotin increases, the ratio of BBP-II to BBP-I increases and becomes constant at dietary intakes of biotin above 250 micrograms/kg. The observation that the amounts of these proteins are limited by biotin in the normal dietary range (less than 250 micrograms/kg) suggests that biotin is required for the synthesis, secretion or stability of these proteins. Although both plasma vitamin-protein complexes are transported to the oocyte and concentrated in the yolk, BBP-II is transferred more efficiently. Thus biotin deposition in the yolk is a function of the amounts and relative concentrations of the two proteins. Dietary biotin above 250 micrograms/kg exceeds the transport capacity of BBP-I and BBP-II in the plasma; however, unbound biotin does not accumulate. Rather it is efficiently scavenged by avidin in the oviduct and transferred to the egg albumen. Only when avidin becomes saturated at high dietary intake does free or weakly bound biotin accumulate in plasma and yolk. The synthesis of avidin is independent of dietary biotin. Small amounts of BBPs with the heat-stability of avidin or BBP-I respectively are present in the plasma of adult males or immature chickens. BBP-II, the major BBP in the plasma and yolk of laying hens, was not detected in the plasma of non-laying chickens.

Avidin Induction by Theophylline in Vivo andin Vitro

Avidin induction in chick tissues in vivo and in vitro was studied by a phosphodiesterase inhibitor, theophylline, and compared to progesterone-dependent induction. Theophylline (100 mg/kg, ip) caused a significant increase in avidin content only in the oviduct of diethylstilbestrol-treated chicks, but not in the lung, muscle, intestine, plasma, or in the bursa of Fabricius. Diethylstilbestrol priming was necessary for oviductal avidin induction in vivo by theophylline. In the oviduct culture, theophylline at a concentration between 100 and 500 micrograms/ml caused a dose-dependent increase in avidin production. Effects of theophylline and progesterone on avidin synthesis in oviduct culture were synergistic. Avidin production was dependent on protein and RNA synthesis, since induction was inhibited by cycloheximide and actinomycin D. Avidin induction by theophylline resembled progesterone-dependent induction, beginning 9 h after the injection in vivo and 12 h after administration of these drugs in vitro. Avidin induced by theophylline showed heat-induced biotin exchange identical to that of progesterone-induced avidin, indicating close similarity of these proteins. The results suggest that theophylline can mimic the action of progesterone on avidin production, and that cyclic nucleotides may have a role in the regulation of avidin synthesis.

Regulation of avidin accumulation by prostaglandins in chick oviduct culture

Regulation of avidin accumulation by prostaglandins (PGs) and their inhibitors was studied in chick oviduct organ culture. Avidin was induced neither by progesterone nor PGF 2α in the oviduct of immature chicks. By progesterone and PGs, a high avidin synthesis was induced when the chicks received diethylstilbestrol (DES) for 7 days. Enhanced avidin production was observed by PGF 2α, PGE 1 and PGE 2, whereas PGA 2 and PGB 2 had a slight inhibitory effect and PGA 1 and PGB 1 had no effect on avidin production. PGF 2α was most effective at a concentration of 10–20 μg/ml. The effects of progesterone and PGF 2α were not additive. Mefenamic acid, at concentrations of 40 and 60 μg/ml, inhibited 50 and 85%, respectively, of the avidin synthesis induced by progesterone, whereas the inhibition of the total protein synthesis was only 20%, and this only by the higher concentration of the drug. Tolfenamic and meclofenamic acid were also inhibitory in the case of progestin-induced avidin synthesis. These studies indicate that the PGs (F 2α, E 1 and E 2) might be involved in the avidin induction in the chick differentiated oviduct. The specific inhibition of the progesterone-dependent avidin synthesis by the PG inhibitors suggests that PGs may be connected with the progesterone action in the oviduct. We propose that the avidin synthesis by the chick oviduct might be considered as a model system for studying PG effects on the synthesis of a specific protein.

Specific Protein Synthesis in Oviduct-progestational Responses of Hens and Oestradiol-primed Chicks: Immunohistochemical Demonstration of Simultaneous Synthesis of Avidin, Trypsin Inhibitor and Ovoinhibitor

Specific Protein Synthesis in Oviduct-progestational Responses of Hens and Oestradiol-primed Chicks: Immunohistochemical Demonstration of Simultaneous Synthesis of Avidin, Trypsin Inhibitor and Ovoinhibitor

Avidin is induced in chicken embryo fibroblasts by viral transformation and cell damage.

Synthesis and secretion of avidin was studied in cultured chicken embryo fibroblasts infected with transforming retroviruses (Rous sarcoma virus, its mutants temperature-sensitive for transformation, OK-10 virus) or a nontransforming retrovirus (RAV-1). Avidin was detectable in both transformed and untransformed cultures, and was identical to chicken egg white avidin by several criteria: biotin-binding, heat-induced biotin exchange, subunit size (mol. wt. 15 600), immunoprecipitation of metabolically labeled proteins and immunoblotting. Transformation increased the production of avidin up to 50-fold, but several experiments suggested that the induction was not a direct consequence of virus-induced cell transformation. The production of avidin seemed to relate to cellular damage both in cultures of virus-transformed and of normal fibroblasts. It may represent a response to cellular damage and viral transformation may activate the process.

Avidin induction by dibutyryl cyclic guanosine 3′,5′-monophosphate in chick oviduct organ culture

A progesterone-dependent protein, avidin, was induced by dibutyryl cGMP in a dose-dependent manner in chick oviduct organ culture. A four-fold concentration of avidin over the control samples was observed with a concentration of dibutyryl cGMP of 1 mM. The time course of avidin accumulation was essentially similar to that caused by progesterone. Dibutyryl cAMP at the same concentration had no effect on the avidin concentration in the cultured oviduct tissue or medium. These results suggest a possible role of cGMP in avidin induction or in the modulation of avidin synthesis.

Characterization of progesterone-independent avidin production of chicken tissues in culture

1. 1. Primary cell cultures of chick oviduct, intestine, peritoneal membrane and lung, but not those of wing muscle or calcaneal tendon, produced avidin progesterone-independently through a 5-week culture. 2. 2. Actinomycin D and cycloheximide, but not hydroxyurea, inhibited the avidin production. 3. 3. The common feature of the cultures producing avidin was a fibroblast-like cell type. 4. 4. Similarly, oviduct and lung, but not muscle or tendon, also produced avidin in hormone-free short-term organ cultures, where the avidin synthesis began during the first 8 h of culture. 5. 5. Induction of the general protein synthesis does not explain the avidin production, since another egg-white protein, ovalbumin, was not induced by the culture procedure.

Age-dependent gene induction in quail oviduct X. Alterations on the post-transcriptional level (enzymic aspect)

In quail oviducts the rate of synthesis of avidin, the biological end-point marker for the molecular events caused by progesterone, decreases with age. The cause of the reduced capacity of avidin induction has been studied on the molecular biological level of polyadenylation of RNA which is one step in the process of post-transcriptional modification of heterogeneous nuclear RNA resulting in the formation of functional mRNA molecules. This novel approach was biochemically possible after the discovery of the poly(A) anabolic enzyme (poly(A) polymerase) and the two poly(A) catabolic enzymes (endoribonuclease IV and 5′-exoribonuclease). These enzymes are involved in the synthesis and degradation of the poly(A) segment of mRNA in vitro and most likely also in poly(A) metabolism in intact cell systems. Enzymatically controlled poly(A) metabolism of mRNA is regulated by the following interrelations: poly(A)-associated proteins and endoribonuclease IV; labilizing factor and poly(A)-associated proteins; 5′-exoribonuclease in cooperation with endoribonuclease IV and poly(A) polymerase. A close correlation between high levels of poly(A) catabolic enzymes and low rate of protein synthesis which was established in cell culture systems, seems also to be partially the biochemical cause for the reduced avidin synthesis in aging quail oviduct.

Age-dependent gene induction in quail oviduct. XI. Alterations on the post-transcriptional level (analytical aspect)

For the elucidation of the age-dependent reduction of avidin synthesis in the oviduct of quails studies on the level of post-transcriptional modification of mRNA were performed. Incorporation of [ 3H]adenosine into mRNA is drastically reduced in senescent animals compared to adult animals. Double labeling of mRNA with [ 3H]adenosine and [ 3H]uridine revealed that in the presence of actinomycin D the incorporation ratio adenosine/uridine into mRNA increases to higher values in the case of adult animals compared with the ratio determined for mRNA from senescent animals. These data support the assumption that in senescent animals the length of the poly(A) sequence on mRNA might be shorter. Because of the low efficiency of label incorporation into poly(A) in whole animals, a method for directly introducing a radioactive label in vitro into the isolated poly(A) was developed for the estimation of the length of the poly(A) segments. A randomly labeled product with no apparent degradation is achieved by methylation of poly(A) with [ 3H]dimethyl sulphate. Therefore the method is a powerful tool for the detection and size determination of small amounts of poly(A). Size determination of poly(A) from oviducts of adult and senescent quails by the methylation method revealed that in the case of senescent animals an increase in shorter poly(A) tracts can be detected. These results support the assumption that the age-dependent reduction of avidin synthesis is also caused by changes in the control mechanisms on the post-transcriptional level.

Progesterone-dependent avidin induction in chick oviduct culture

A study was made of the factors influencing progesterone-dependent induction of oviductal avidin in organ culture. Expiants of oviductal tissue from immature estrogen-pretreated chicks were cultured in vitro. Avidin was assayed by [ 14C]-biotin binding and RIA method, and its synthesis by amino acid incorporation. Estrogen priming for 7 days was necessary for a maximal avidin response. Induction with progesterone in the medium for 24 h promoted a dose-related increase in avidin content in medium and tissue. A high concentration of progesterone (50 μg/ml) was deleterious to avidin production. Avidin synthesis began 8–10 h after progesterone administration. A full avidin response was obtained when progesterone was present in the medium only during the first hour of a 24-h incubation, which indicates that the continued presence of progesterone in the medium is not required for avidin synthesis. Control oviducts constantly produced avidin, apparently induced by tissue trauma during the sample preparation. Ethanol reduced progesterone-independent avidin but had no effect on progesterone-dependent avidin. The major part of the avidin produced was secreted into the medium. Avidin induction was inhibited by cycloheximide (10 μg/ml) and NaF (4mM). This was due to the inhibition of avidin synthesis by these drugs. The effect of NaF was reversible. In conclusion, progester-one-dependent and -independent avidin synthesis in vitro is described, both being dependent on new protein synthesis.

Avidin synthesis in vitro by chick oviduct after a short induction with progesterone or actinomycin D in vivo

The continuation of avidin synthesis in the chick oviduct was studied after a short progesterone or actinomycin D induction. Diethylstilboestrol pretreated chicks received a single injection of progesterone or actinomycin D. Chicks were killed 4, 6, 9, 12, 16 or 24 h after treatment. Pieces of oviducts were assayed directly for avidin or incubated at 40°C in culture medium. AH incubations were terminated 24 h after the injection. Avidin from tissue and medium was measured separately. Avidin induction could not be transferred to the culture system if the exposure to progesterone in vivo was less than 6 h. When the induction time in vivo was 6h, the tissue avidin concentration increased during subsequent incubation from 0.04 to 9.2 μg/g tissue. No more increase in tissue avidin concentration was found in samples obtained from chicks 12 h after progesterone during the following incubation for 12h. Progesterone in the incubation medium did not cause any further increase in the final avidin amount. Actinomycin D. administered in vivo, caused an increase in avidin concentration during incubation similar to that in control tissues. In conclusion, progesterone causes avidin production which continues even when it is removed. On the other hand, no such “switch on” induction occurs with actinomycin D. It is therefore unlikely that progesterone mediates avidin induction caused by actinomycin D. which would indicate that there is both progesterone-dependent and -independent avidin induction in the chick oviduct.

Induction of avidin in the chick oviduct by tissue damage and prostaglandins.

In immature, diethylstilboestrol-treated chicks, ligation of the oviduct caused local avidin synthesis in the immediate vicinity of the ligature. PGF-2alpha injected directly into the oviduct also induced avidin synthesis, whereas saline or PGE-2 had no effect. PGE and PGF-2alpha concentrations increased in the oviduct within 24 h of ligation: the PGE increase could be partly inhibited by indomethacin, whereas that of PGF-2alpha was less inhibited. An LD50 dose of indomethacin alone and with ligation had a clear stimulatory effect on avidin synthesis, whereas aspirin alone, or with ligation, was not effective. Ligation alone and with indomethacin appeared to alter the PGF-2alpha/PGE ratio. These results suggest that PGF-2alpha may be involved in the regulation of avidin synthesis in the chick oviduct.

INDUCTION OF AVIDIN SYNTHESIS BY RNA OBTAINED FROM LIZARD OVIDUCTS

Instituto di Zoologia, Università di Napoli, Via Mezzocannone 8, Napoli, Italy (Received 31 December 1976) In the lizard Lacerta s. sicula sex steroids induce the synthesis of avidin in the oviduct in vivo (Botte, Segal & Koide, 1974). We report here evidence that these effects could be mediated, in vivo and in vitro, by RNA. Adult female Lacerta s. sicula were maintained in terraria at 28 °C, and under a natural photoperiod. The lizards were ovariectomized and used 15 days after the operation. For the RNA preparation, three groups of ovariectomized females, each comprising 40 animals, were treated as follows: Group 1, four doses of 50 μg oestradiol dipropionate in 0·05 ml almond oil (every 3 days), administered intramuscularly; Group 2, treated with oestradiol as in Group 1, but only three times; on the fourth treatment given 0·5 mg progesterone in 0·05 ml almond oil; Group 3, injected with almond

Effect of progesterone on HnRNA and avidin mRNA of the chick oviduct

Nuclear pre-mRNA (HnRNA) and polysomal avidin mRNA were extracted from diethylstilbestrol and progesterone-treated immature chick oviducts. Purity of avidin antibody precipitated polysomes as well as isolated nuclei were confirmed by electron microscopy (EM). The number of perichromatin granules was counted from the oviductal epithelial cell nuclei. Form and size of RNA extracts were studied with EM. Avidin coding activity of RNA's was determined in a rabbit reticulocyte lysate system. The nuclei and polysomes of the control animals contained the same amount of avidin coding activity per 5 g of the oviduct tissue. During the progesterone induction of the avidin synthesis the activity of nuclear pre-mRNA coding for avidin decreases while the cytoplasmic avidin mRNA increases. The total avidin coding activity increases only slightly during 24 h after progesterone. There is an initial decrease in the amount of perichromatin granules followed by an increase exceeding the level of the controls 12 and 24 h after progesterone. The EM analysis of RNA extracts revealed that the average size of pre-mRNA molecules was 12000–13000 nucleotides, whereas the size of the mRNA molecules was considerably smaller, the major portion of the mRNA fraction was about 3000 nucleotides in length. The present results suggest that the cleavage and transfer of the nuclear pre-mRNA might be the critical steps of the non-transcriptional control of avidin synthesis by progesterone.

Induction of avidin in the chick oviduct by tissue damage. Effect of promethazine chloride, CaCl 2 and hydrocortisone on local induction

The local effect of the mechanical induction of avidin by ligature was studied in diethylstilbestrol-primed chicks. The highest induction of avidin was always found in the immediate vicinity of the silk ligature of the oviduct. The locality of the induction was highly dependent on the position of the ligature. The nonligated parts of the ligated oviduct also showed a slight avidin induction. These results indicate a strictly local effect of avidin induction by ligature. An antihistamine, promethazine chloride, has a potentiating effect on the avidin induction by ligature when administered after the ligature. On the other hand, membrane stabilization by hydrocortisone or CaCl 2 did not influence the ligature-induced avidin synthesis. On the basis of these results it is concluded that the avidin induction is not mediated by histamine activation or membrane damage.

TRANSLATION OF AVIDIN mRNA FROM CHICK OVIDUCT IN THE OESTROUS MOUSE UTERUS

Oestrogen-primed immature chicks were injected with 5.0 mg of progesterone. Twenty hours after progesterone avidin mRNA was isolated and purified by a method including an antibody precipitation of the avidin polysomes, phenol extraction and nitrocellulase trapping. A water solution of mRNA (1 mug) with [14C]amino acids was instilled into the uterine horn of oestrous or dioestrous mice. Saline and [14C]amino acids were injected into the opposite control uterine horn. Avidin was assayed by an antibody method. A clear-cut avidin synthesis was found in the oestrous uterus, whereas the dioestrous showed only a slight avidin synthesis. This suggest that there are no absolute species-specific factors required at the translational level of uterine protein synthesis and that the mammalian uterus can translate foreign hormone-specific proteins at a rate dependent on the endogenous protein synthesis.

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.