Rooster Liver Research Articles

Identification and characterization of long noncoding RNAs and mRNAs expression profiles related to postnatal liver maturation of breeder roosters using Ribo-zero RNA sequencing

BackgroundThe liver is mainly hematopoietic in the embryo, and converts into a major metabolic organ in the adult. Therefore, it is intensively remodeled after birth to adapt and perform adult functions. Long non-coding RNAs (lncRNAs) are involved in organ development and cell differentiation, likely they have potential roles in regulating postnatal liver development. Herein, in order to understand the roles of lncRNAs in postnatal liver maturation, we analyzed the lncRNAs and mRNAs expression profiles in immature and mature livers from one-day-old and adult (40 weeks of age) breeder roosters by Ribo-Zero RNA-Sequencing.ResultsAround 21,939 protein-coding genes and 2220 predicted lncRNAs were expressed in livers of breeder roosters. Compared to protein-coding genes, the identified chicken lncRNAs shared fewer exons, shorter transcript length, and significantly lower expression levels. Notably, in comparison between the livers of newborn and adult breeder roosters, a total of 1570 mRNAs and 214 lncRNAs were differentially expressed with the criteria of log2fold change > 1 or < − 1 and P values < 0.05, which were validated by qPCR using randomly selected five mRNAs and five lncRNAs. Further GO and KEGG analyses have revealed that the differentially expressed mRNAs were involved in the hepatic metabolic and immune functional changes, as well as some biological processes and pathways including cell proliferation, apoptotic and cell cycle that are implicated in the development of liver. We also investigated the cis- and trans- regulatory effects of differentially expressed lncRNAs on its target genes. GO and KEGG analyses indicated that these lncRNAs had their neighbor protein coding genes and trans-regulated genes associated with adapting of adult hepatic functions, as well as some pathways involved in liver development, such as cell cycle pathway, Notch signaling pathway, Hedgehog signaling pathway, and Wnt signaling pathway.ConclusionsThis study provides a catalog of mRNAs and lncRNAs related to postnatal liver maturation of chicken, and will contribute to a fuller understanding of biological processes or signaling pathways involved in significant functional transition during postnatal liver development that differentially expressed genes and lncRNAs could take part in.

Selective Estrogen Receptor Modulators Tamoxifen, Pterostilbene, Raloxifene and Clomiphene fail to stimulate the Hepatic Expression of Estrogen Responsive Genes Encoding the Avian Apolipoprotein II and Vitellogenin

Apolipoprotein (apo) II and vitellogenin (VTG) I, II &amp; III are yolk proteins expressed in the avian liver in response to estrogen during egg‐laying. Estrogenic stimulation of the hepatic expression of apo II and VTG is due to both transcriptional increase and mRNA stabilization. We had previously shown the expression of the estrogen‐responsive mRNA stabilizing factor, which stabilizes apo II mRNA to be stimulated by estrogen, and selective estrogen receptor modulators (SERMs) such as genistein, resveratrol, tamoxifen, Aroclor, chlordecone (Kepone) and hexachlorophene, but inhibited by clomiphene, toxaphene and bisphenol‐A. Reverse Transcription‐Polymerase Chain Reaction (RT‐PCR), Northern blot analysis, RNase protection assays on whole cell RNA and Western blot analysis of cytosolic extracts from livers of roosters treated with estrogen or SERMs such as tamoxifen, pterostilbene, raloxifene and clomiphene showed stimulation of transcription and expression of apo II and VTG I, II &amp; III by estrogen but not the SERMs, demonstrating selective regulation of estrogen‐responsive genes by the latter. This work was supported by science research funds from Arnold and Marie Schwartz College of Pharmacy, Long Island University, NY.

Selective Estrogen‐Receptor Modulators Genistein, Resveratrol, and Catechin fail to stimulate the Hepatic Expression of Estrogen‐Responsive Genes Encoding the Avian Apolipoprotein II and Vitellogenin

Apolipoprotein (apo) II and vitellogenin (VTG) I, II &amp; III are yolk proteins expressed in the avian liver in response to estrogen during egg‐laying. Estrogenic stimulation of the hepatic expression of apo II and VTG is due to both transcriptional increase and mRNA stabilization. We had previously shown the estrogen‐responsive mRNA stabilizing factor, which stabilizes apo II mRNA to be stimulated by estrogen, and selective estrogen receptor modulators (SERMs) such as genistein, resveratrol, tamoxifen, Aroclor, chlordecone (Kepone) and hexachlorophene, but inhibited by clomiphene, toxaphene and bisphenol‐A (Ratnasabapathy et al. 53:1425–1434, 1997, Ratna and Oyemalu, C. J. Steroid Biochem. Mol Biol. 80:383–393, 2002, Ratna, W.N. &amp; Simonelli, Life Sci. 70:1577–1589, 2002; Ratna, W.N. Life Sci 71:865–877, 2002). Reverse Transcription‐Polymerase Chain Reaction (RT‐PCR), Northern blot analysis, RNase protection assays on whole cell RNA and Western blot analysis of cytosolic extracts from livers of roosters treated with estrogen or SERMs such as genistein, resveratrol and catechin showed stimulation of transcription and expression of apo II and VTG I, II &amp; III by estrogen but not the SERMs, demonstrating selective regulation of estrogen‐responsive genes by the latter. This work was supported by science research funds from Arnold and Marie Schwartz College of Pharmacy, Long Island University, NY.

Enzyme profile of heart and skeletal muscles, liver and lung of roosters and geese.

Summary Enzymes profiles of liver, lung, heart and skeletal muscles from geese and roosters are described, based on the activity of glutamate-oxalacetate transaminase, glutamate-pyruvate transaminase, lactate dehydrogenase, alkaline phosphatase, cholinesterase, and isocitrate dehydrogenase. The importance and potential of knowing the enzyme profile in veterinary clinical biochemistry is discussed. Zusammenfassung Enzymprofile von Herz und Skelettmuskel sowie von Leber und Lunge von Hähnen und Gänsen Basierend auf der Aktivitít der Glutamat-Oxalacetat Transaminase, der Glutamat-Pyruvat Transaminase, der Laktatdehydrogenase, der alkalischen Phosphatase, der Cholinesterase und der Isozitratdehydrogenase wurden die Enzymprofile von Leber, Lunge, Herz und Skelettmuskeln von Gänsen und Hähnen bestimmt. Die klinische Bedeutung solcher Enzymprofile wird besprochen. Résumé Profils enzymatiques du muscle squelettique et cardiaque ainsi que du foie et des poumons chez des coqs et des oies Les profils enzymatiques du foie, des poumons, du muscle squelettique et cardiaque d'oies et de cogs ont été déterminés sur la basse de l'activité de la transaminase, glutamo-oxalacétique, de la transaminase glutamo-pyruvique, de la lactino-dehydrogénase, de la phosphatase alcaline, de la cholinestérase et de 'lisocitrate-dehydrogénase. On discute la signification clinique de ces profils enzymatiques. Resumen Perfiles enzimáticos del corazón, músculos esqueléticos, hígado y pulmón de pollos y gansos Basándose en las actividades de la transaminasa glutamato-oxalacetato, transminasa glutamato-piruvato, lactatodehidrogenasa, fosfatasa alcalina, colinesterasa e isocitratodehidrogenasa, se valoraron los perfiles enzimáticos de hígado, pulmón, corazón y músculos esqueléticos de gansos y gallos. Se discute la importancia y potencial de conocimientos en bioquímica veterinaria clínica de estos perfiles de fermentos.

Identification and characterization of phosphoseryl-tRNA[Ser]Sec kinase.

In 1970, a kinase activity that phosphorylated a minor species of seryl-tRNA to form phosphoseryl-tRNA was found in rooster liver [Maenpaa, P. H. & Bernfield, M. R. (1970) Proc. Natl. Acad. Sci. USA 67, 688-695], and a minor seryl-tRNA that decoded the nonsense UGA was detected in bovine liver. The phosphoseryl-tRNA and the minor UGA-decoding seryl-tRNA were subsequently identified as selenocysteine (Sec) tRNA[Ser]Sec, but the kinase activity remained elusive. Herein, by using a comparative genomics approach that searched completely sequenced archaeal genomes for a kinase-like protein with a pattern of occurrence similar to that of components of Sec insertion machinery, we detected a candidate gene for mammalian phosphoseryl-tRNA[Ser]Sec kinase (pstk). Mouse pstk was cloned, and the gene product (PSTK) was expressed and characterized. PSTK specifically phosphorylated the seryl moiety on seryl-tRNA[Ser]Sec and, in addition, had a requirement for ATP and Mg2+. Proteins with homology to mammalian PSTK occur in Drosophila, Caenorhabditis elegans, Methanopyrus kandleri, and Methanococcus jannaschii, suggesting a conservation of its function across archaea and eukaryotes that synthesize selenoproteins and the absence of this function in bacteria, plants, and yeast. The fact that PSTK has been highly conserved in evolution suggests that it plays an important role in selenoprotein biosynthesis and/or regulation.

The Major Chicken Egg Envelope Protein ZP1 Is Different from ZPB and Is Synthesized in the Liver

The extracellular matrix surrounding vertebrate oocytes is called the zona pellucida in mammals and perivitelline membrane (pvm) in birds. We have analyzed this structure in chicken follicles and laid eggs and have identified a 95-kDa component of the pvm, which, by protein sequencing, shows homology to mammalian zona pellucida proteins. Surprisingly, we could not detect this protein in ovarian granulosa cells or oocytes but instead found high levels in the liver of the laying hen. In contrast, it is absent in rooster liver but can be efficiently induced by estrogen treatment of the animal. An immunoscreen of a liver lambda-ZAP library yielded a cDNA coding for a protein of 934 amino acids. It displayed significant homology to members of the ZP1/ZPB family from other species, notably to mouse and rat ZP1, and was therefore designated chkZP1. It is clearly different from a protein designated chkZPB that had been deposited in the data base previously. Alignment of the known members of the ZP1/ZPB family demonstrated the existence of at least three subgroups, with representatives of both the ZP1 and the ZPB sequence homology group occurring in vertebrates. Northern blot analysis of liver extracts revealed the presence of a single 3. 2-kilobase mRNA coding for chkZP1, distinct from the chkZPB transcript detectable in follicles. Immunohistochemical analysis of follicle sections demonstrates that chkZP1 can be found in the blood vessels of the theca cell layer as well as in the pvm surrounding the oocyte. Thus, in the chicken, at least one of the major pvm components is synthesized in the liver and is transported via the bloodstream to the follicle.

In vivo estradiol-dependent dephosphorylation of the repressor MDBP-2-H1 correlates with the loss of in vitro preferential binding to methylated DNA.

We have previously shown that estradiol treatment of roosters resulted in a rapid loss of binding activity of the repressor MDBP-2-H1 (a member of the histone H1 family) to methylated DNA that was not due to a decrease in MDBP-2-H1 concentration. Here we demonstrate that MDBP-2-H1 from rooster liver nuclear extracts is a phosphoprotein. Phosphoamino acid analysis reveals that the phosphorylation occurs exclusively on serine residues. Two-dimensional gel electrophoresis and tryptic phosphopeptide analysis show that MDBP-2-H1 is phosphorylated at several sites. Treatment of roosters with estradiol triggers a dephosphorylation of at least two sites in the protein. Phosphatase treatment of purified rooster MDBP-2-H1 combined with gel mobility shift assay indicates that phosphorylation of MDBP-2-H1 is essential for the binding to methylated DNA and that the dephosphorylation can occur on the protein bound to methylated DNA causing its release from DNA. Thus, these results suggest that in vivo modification of the phosphorylation status of MDBP-2-H1 caused by estradiol treatment may be a key step for the down regulation of its binding to methylated DNA.

Characterization of liver-enriched proteins binding to a developmentally demethylated site flanking the avian apoVLDLII gene.

Although the avian apoVLDLII gene is normally expressed exclusively in the liver of the laying hen, the gene can be activated by estrogen in birds of either sex beginning between days 7-9 of embryogenesis. Developmentally programmed demethylation of sites in the 5'- and 3'-flanking regions of the gene have been shown to occur during this period of embryogenesis, suggesting that they may reflect changes in protein-DNA interactions that are involved in the acquisition of competence to activate the apoVLDLII gene. We have detected specific protein interactions at one location approximately 2.6 kb upstream from the apoVLDLII gene, that includes an Msp I site whose methylation status changes between days 7 and 9 of embryogenesis. The sequence of this region bears significant similarity to binding sites of members of the bZIP family of liver-enriched or -specific factors such as C/EBP, DBP, and LAP, that are characteristically produced relatively late during liver development. In the studies described here, we demonstrate that proteins binding to the upstream apoVLDLII site do not correspond to previously identified liver-enriched or -specific factors. They also display a pattern of activity during development and in human and avian hepatoma cell lines indicating that their expression is increased in proliferating cells. Southwestern blotting and UV cross-linking studies indicate that two proteins of approximately 60 kD are capable of binding to the site and we describe the purification of these factors from crude nuclear protein extracts obtained from rooster liver.

Proteins associated with the messenger ribonucleoprotein particle for the estrogen-regulated apolipoprotein II mRNA

The stability of the mRNA for apolipoprotein (apo) II is regulated by estrogen [Gordon et al. (1988) J. Biol. Chem. 263, 2625-2631]. On the hypothesis tha estrogen regulation of apoII mRNA stability is mediated through mRNA-protein interaction, we have examined the messenger ribonucleoprotein particle (mRNP) for apoII mRNA following release from chicken liver polyribosomes. Polyribosomes containing undegraded apoII mRNA were obtained when tissue was homogenized without detergent, and polyribosomes were isolated following simultaneous addition of detergent and magnesium to a 20000g supernatant. ApoII mRNP released by EDTA sedimented at 12-18 S in sucrose gradients, and banded at rho = 1.4 g/mL in CsCl isopycnic centrifugation, indicative of a 3:1 ratio of protein to mRNA. A fraction in which apoII mRNP was enriched to 40-50% of total mRNP was prepared by successive size fractionation steps on sucrose gradients. Proteins associated with sucrose gradient enriched apoII mRNP were examined by iodination of UV-cross-linked proteins followed by SDS-polyacrylamide gel electrophoresis. Comparisons of proteins in highly enriched apoII mRNP to proteins in mRNP from non-estrogen-treated rooster liver did not reveal any differences. This result suggests that the major proteins associated with apoII mRNA are mRNP proteins also associated with the bulk of liver mRNAs.

14] Choline/ethanolamine kinase from rat kidney

Choline kinase (ATP:choline phosphotransferase) and ethanolamine kinase (ATP:ethanolamine O-phosphotransferase) catalyze the phosphorylation of choline/ethanolamine by ATP in the presence of Mg 2+ , yielding phosphocholine/phosphoethanolamine and ADP. This enzyme step commits choline (ethanolamine) to the CDPcholine (CDPethanolamine) pathway for the biosynthesis of phosphatidylcholine (phosphatidylethanolamine) in all animal cells. The chapter discusses different methods to assay choline kinase activity. The purification of choline/ethanolamine kinase from rat kidney is also discussed. Purification is started with the high-speed supernatant fraction from fresh kidneys (∼200 g wet weight). All procedures are carried out below 5 ° C. One of the most important features of choline/ethanolamine kinase is its inducibility in various experimental systems. Although the physiological significance of the inducibility has not yet been fully understood, the induction of choline/ethanolamine kinase by several means suggests that more than one mechanism could be involved in this process. Distinct mechanisms of choline kinase induction have been demonstrated between rooster liver by estrogens and rat liver by certain hepatotoxins. A new isozyme of choline/ethanolamine kinase appears to be preferentially induced in the latter case.

In vivo and in vitro protein-DNA interactions at the distal oestrogen response element of the chicken vitellogenin gene: Evidence for the sane protein binding to this sequence in hen and rooster liver

The major egg white protein, vitellogenin, is synthesized in a tissue specific and oestradiol dependent manner in the liver of egg-laying hens. In this paper, we describe a detailed study of the protein-DNA interactions at the distal oestrogen response element (ERE D) located 600 bp upstream of the start of transcription. In vivo footprinting of hepatocytes from adult hens and roosters with 0.5–0.0005% dimethylsulphate (DMS) revealed, at critical concentrations of DMS, protection of distinct guanosine residues within the ERE D and adjacent downstream sequence in both cases. From this, it was concluded that there were proteins present in both tissues binding to this region in vivo. In vitro studies using missing base contact probing and proteolytic clipping band shift assays wuth hen and rooster liver nuclear extracts identified the ERE binding protein to be the same of very closely related in both tissues. Furthermore, the proteon from rooster nuclear extracts bound to the ERE sequence even when the DNA was methylated at CpG dinucleotides. u.v. cross-linking experiments performed with bromodeoxyuridine substituted ERE, revealed that a nuclear protein with M r of about 75,000–80,000 bound specifically to this sequence. These studies demonstrate that apart from the oestrogen receptor, at least one other protein can interact specifically with the chicken vitellogenin ERE, independently of hormonal expression of the gene.

Estradiol down regulates the binding activity of an avian vitellogenin gene repressor (MDBP-2) and triggers a gradual demethylation of the mCpG pair of its DNA binding site

A negative regulating protein (MDBP-2) from rooster liver nuclear extracts binds preferentially to a methylated promoter region 5'TTCACCTTmCGCTATGAGGGGGATCATACTGG3' of the avian vitellogenin II gene (Nucleic Acids Res. 19, 1029-1034, 1991). Treatment of adult and immature roosters with estradiol results in a 90% decrease in the binding activity of MDBP-2 within three days. This corresponds to the level found in egg laying hens. The decrease in the binding activity of MDBP-2 precedes the onset of vitellogenin gene transcription. At the same time, there is a two-fold increase in the binding activity of NHP-1 (tested with the same oligonucleotide as for MDBP-2), a protein thought to be involved in the active demethylation of DNA. The methylated oligonucleotide binds either MDBP-2 or NHP-1 and there is no complex formation between the two proteins and DNA. Estradiol treatment does not change the equilibrium binding constant of MDBP-2 which is about 10(-9)M for the methylated oligonucleotide. The early kinetics of demethylation of the mCpG pair in the binding site of MDBP-2 was studied by means of genomic sequencing. A low level of demethylation of mCpG starts gradually on both DNA strands already 4 hours after estradiol treatment during the lag phase of vitellogenin mRNA synthesis. It is concluded that the lowering of the binding activity of MDBP-2 may have a stronger effect on the derepression of the gene than the slow demethylation of MDBP-2 DNA binding site. The role of the methylated CpG is to assure a high binding affinity of the repressor to DNA.

Positive and negative regulatory elements of chicken vitellogenin II gene characterized by in vitro transcription competition assays in a homologous system.

A homologous in vitro transcription system was developed in which the cloned chicken vitellogenin II gene is faithfully transcribed by extracts prepared from chicken liver nuclei. The use of template deleted of its upstream region resulted in poor transcriptional efficiency, as did the use of extracts prepared from rooster liver, in which the gene is silent. The influence of individual cis elements was determined by transcription competition analysis. Oligonucleotides covering greater than 500 base pairs of the promoter region were used as competitor DNA in the in vitro reactions. Competition with an oligonucleotide covering part of the expression-specific DNase I hypersensitivity site B2, which contains a demethylation site, mCpG, at nucleotide position + 10, increased transcription of the gene, suggesting the binding of a repressor to this region. The enhancement of transcription was even more pronounced when the same oligonucleotide was methylated at the corresponding + 10 cytosine. Competition with oligonucleotides covering the TATA box, or the estrogen response element half-palindromic motif (GGTCA) at nucleotide positions -198 to -194, resulted in a large decrease in vitellogenin gene transcription, indicating that strongly activating factors bind to these regions. Competing oligonucleotides covering other GGTCA-containing motifs situated further upstream at nucleotide positions -292 to -288, -367 to -351, and -626 to -614 were increasingly less effective in inhibiting transcription. The results indicate that factors other than the estrogen receptor are involved in transcriptional activation of the vitellogenin II gene.

Expression of Endogenous and Transfected Apolipoprotein II and Vitellogenin II Genes in an Estrogen Responsive Chicken Liver Cell Line

A recently described chicken liver cell line, LMH, was characterized to evaluate responsiveness to estrogen. Expression of the endogenous apolipoprotein (apo) II gene was induced by 17 beta-estradiol when LMH cells were cultured with chicken serum. The response was low and yielded apoll mRNA at only 0.3% of the level seen in estrogenized rooster liver. Higher levels of apoll mRNA were achieved when LMH cells were transiently transfected with an expression plasmid for estrogen receptor. A transfected apoll gene was strongly expressed only when cotransfected with receptor. Expression of the endogenous vitellogenin (VTG) II gene was not detected. However, when cotransfected with a receptor expression plasmid, VTG II reporter plasmids were expressed in LMH cells in response to 17 beta-estradiol. These results suggest that estrogen responsiveness of LMH cells is limited by the availability of functional receptor. Low levels of estrogen receptor mRNA were detected in LMH cells, and receptor binding sites and mRNA were greatly increased following transient transfection with a receptor expression plasmid. Using this transient transfection protocol, several VTG II reporter plasmids were compared in LMH cells and chick embryo fibroblasts. A plasmid containing VTG II estrogen response elements linked to a heterologous promoter was regulated by estrogen in both cell types. In contrast, reporter plasmids containing the VTG II promoter were regulated by estrogen in LMH cells but were not expressed at all in chick embryo fibroblasts. These results suggest that regulation of the VTG II gene involves cell type-specific elements in addition to estrogen response elements.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and analysis of nuclear bodies from estrogen-stimulated chick liver

The target cells of most steroid hormones contain prominent structures termed nuclear bodies (NB). Estrogen (ES) responsive tissues have very distinctive NB, whose development has been associated with both the level of ES receptors and their retention times in nuclei. It has been suggested that these NB may be large-scale ES binding centers at transcriptionally hyperstimulated sites. We examined the formation of NB in ES-stimulated rooster liver, after primary induction of vitellogenesis and during subsequent decay of this response. Distinct NB appear 6 h postinduction, initially as compact spheroids with ring-like profiles and later on as more complex morphotypes. Nuclei average 15–17 NB each at peak induction (24–48 h), but this declines again gradually as the hormonal response abates. This transient pattern is paralleled closely by the [ 3H]ES binding capacity of nuclei in vitro and the calculated number of specific hormone receptors present. There is no direct numerical correspondence, however, between the NB and the ES receptors, either on a one-to-one basis or as some simple ratio. Hence any links between ES binding activity in nuclei and the NB must be both complex and indirect. Nuclear bodies were isolated following lysis of nuclei by sonication and separation of subfractions on discontinuous sucrose gradients. The resultant NB-enriched fraction consisted primarily of protein, with traces of DNA and RNA. Analysis of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a complex pattern of polypeptides, some apparently unique and probably NB-derived.

Genomic sequencing and in vivo footprinting of an expression-specific DNase I-hypersensitive site of avian vitellogenin II promoter reveal a demethylation of a mCpG and a change in specific interactions of proteins with DNA.

Genomic sequencing was used to study the in vivo methylation pattern of two CpG sites in the promoter region of the avian vitellogenin gene. The CpG at position +10 was fully methylated in DNA isolated from tissues that do not express the gene but was unmethylated in the liver of mature hens and estradiol-treated roosters. In the latter tissue, this site became demethylated and DNase I hypersensitive after estradiol treatment. A second CpG (position -52) was unmethylated in all tissues examined. In vivo genomic footprinting with dimethyl sulfate revealed different patterns of DNA protection in silent and expressed genes. In rooster liver cells, at least 10 base pairs of DNA, including the methylated CpG, were protected by protein(s). Gel-shift assays indicated that a protein factor, present in rooster liver nuclear extract, bound at this site only when it was methylated. In hen liver cells, the same unmethylated CpG lies within a protected region of approximately equal to 20 base pairs. In vitro DNase I protection and gel-shift assays indicate that this sequence is bound by a protein, which binds both double- and single-stranded DNA. For the latter substrate, this factor was shown to bind solely the noncoding (i.e., mRNA-like) strand.

Protein-DNA interactions in vitro with 5′-flanking dna fragments from the chicken vitellogenin gene

The expression of the vitellogenin gene in the liver of oviparous animals is under strict control of estrogen. We have studied the interaction of proteins extracted from nuclei of different estrogen responsive tissues with two fragments (−728 to −470 and −625 to −470) of the upstream region of the chicken vitellogenin gene, using the gel-retardation technique. We found a complex pattern of retarded bands using nuclear extracts from laying hen liver, rooster liver and MCF-7 cells. The patterns observed display differences in the position and intensities of some of the bands, depending on the source of the extract used. The possible significance of these findings will be discussed.

Turnover products of the apo very low density lipoprotein II messenger RNA from chicken liver.

The mature apo Very Low Density Lipoprotein II (apo VLDLII) mRNA appears in chicken liver within a few hours after estrogen administration. Apart from this mRNA species, shorter RNA molecules hybridizing to apo VLDLII sequences have been detected in rooster liver upon estrogen stimulation. These molecules are present in the non-polyadenylated fraction of the total cellular- and polysomal RNA. Northern blotting and electron microscopy of R-loops were employed to show that these shorter RNA molecules are truncated at their 3'-end. The 3'-termini were further characterized by nuclease S1 analyses, and are located predominantly in the 3' untranslated region of the mRNA. Using a secondary structure model (Shelness and Williams, J. Biol. Chem. 260, 8637-8646, 1985), we show that the 3' termini map mainly in unpaired regions of the structure.

Redifferentiation and subsequent dedifferentiation of the livers of roosters resulting from acute estrogen challenges

1. l. Histologic and metabolic changes take place in livers of rooster receiving challenges consisting of acute doses of estrone. 2. 2. During initial Growth and Redifferentiation livers rapidly increase in size by division of hepatocytes within most lobules, changing from cordlike to acinar configurations. 3. 3. No new lobules appear and degeneration of some cells within lobules takes place even as cell divisions predominate. 4. 4. Cells within lobules assume secretory features. 5. 5. Vitellogenins, very low density lipoproteins, calcium and alkaline phosphatase increase greatly in plasma. 6. 6. Within 35–40 days of cessation of estrogen, livers have returned to near normal sizes and plasmas exhibit normal parameters.

Differential estrogen responsiveness of the vitellogenin and apo very low density lipoprotein II genes in the rooster liver

The primary transcript of the chicken apo Very Low Density Lipoprotein II (apoVLDL-II) gene is formed almost immediately after a first estrogen administration, contrary to the appearance of the vitellogenin primary transcript which has a lag of at least 4 h. However, after a second estrogen administration the vitellogenin gene transcription shows no detectable lag (memory effect). After estrogen withdrawal, the primary transcripts of both genes rapidly decline to undetectably low levels. In the presence of estrogen, the half-lives of the mRNAs of apoVLDL-II and vitellogenin are 15 and at least 70 h, respectively, whereas in the absence of hormone they are only 3.5 and 5.5 h, respectively. Thus estrogen not only controls the transcription of both genes, but also the turnover of their mRNAs. In short, there appears to be a quantitative difference in the response of both genes.