Androgenic regulation of rapidly synthesized RNA in the rat ventral prostate.

The influence of castration on the incorporation of nucleotide precursors into RNA of isolated prostatic tissue has been investigated. Castration brought about an apparent increase of incorporation by increasing the specific activity of the uridine nucleotide pool and not by an enhancement in synthesis of rapidly labelled RNA. An actual decrease in this reaction was shown by dividing the radioactivity incorporated into RNA by the specific activity of the uridine nucleotide. Administration of testosterone brought about enhanced synthesis of RNA in the prostates of rats castrated for 3 days as early as 4 h after injection, reaching maximum effect at 8 h. No significant difference was found between the turnover rates of rapidly labelled RNA from castrated, androgen-treated castrated and intact rats. The increased prostatic permeability to nucleosides as an early action of androgens in this organ is discussed.     

Testosterone induction of poly(A)(+)-RNA synthesis and [35S]methionine incorporation into proteins of Rana esculenta Harderian gland.

The role of androgens in the cyclic secretory activity of the Rana esculenta Harderian gland (HG) was studied. Total RNA showed a dramatic increase in October and May when the nuclear androgen receptors peak. During the resumption of the secretory activity a gradual increase of poly(A)(+)-RNA was detected; during the enhancement phase (May) a peak of the poly(A)(+)-RNA fraction was found. In in vitro experiments testosterone increased the incorporation of [3H]uridine into the poly(A)(+)-RNA fraction and also that of [35S]methionine into a newly synthesized protein fraction (100 kDa). The latter effect is prevented by the exposure of the cells to the antiandrogen, cyproterone acetate (CPA). These findings reveal that, besides hamsters, the HG is a target for androgens in the frog.     

Hormone-induced increase in levels of functional mRNA and alpha-amylase mRNA in barley aleurones

Incubation of barley aleurone cells with gibberellic acid produces a progressive increase in the RNA content of the cells. The activity of poly(A)-containing RNA, measured in an in vitro wheat germ protein-synthesizing system, reaches a maximum ≈12 hr after hormone addition and declines thereafter. The structurally intact functional mRNA content in these cells, measured as poly(A)-RNA with 5′ “caps,” also shows a maximum at 12 hr and correlates with the translational capacity of poly(A)-RNA. Activation of mRNA by guanylylation or methylation after addition of gibberellic acid is ruled out. Available evidence indicates that gibberellic acid stimulates protein synthesis by increasing the synthesis of mRNA. Studies with cycloheximide suggest that the induction of synthesis of α-amylase mRNA by gibberellic acid requires protein synthesis after hormone addition.     

Androgen regulation of canine prostatic arginine esterase mRNA using cloned cDNA

Canine prostatic arginine esterase complementary DNA has been cloned in pPBS27, a new cloning vector. The relative abundance of androgen-regulated mRNA in intact dog prostate was reflected by the finding that a high proportion of the clones in the cDNA library hybridized strongly by plaque or colony hybridization with a poly(A)+ RNA probe from intact dog prostate but not with a poly(A)+ RNA probe from castrated dog prostate. One clone carrying a 400 base pairs cDNA insert was selected for further studies. Translation of the hybrid-selected RNA in a cell-free system resulted in the production of a 31 kDa peptide immunoprecipitable by antibodies against arginine esterase. This identification was confirmed by partial sequence analysis of the cDNA revealing an encoding protein with high homology to known kallikreins. Northern blot analysis of poly(A)+ and total RNA showed that arginine esterase mRNA had an approximate size of 1.0 kb which corresponded to a major androgen-regulated RNA species that could be observed after denaturing agarose gel electrophoresis of prostatic poly(A)+ RNA from intact dogs. Dot-blot analysis showed that dogs which had been castrated 3 weeks before had more than 100-fold lower arginine esterase mRNA level than intact dogs or castrated dogs treated with Depo-testosterone.     

Interference of nonsense mutations with eukaryotic messenger RNA stability.

The fine structure map of the yeast URA 3 gene was established by meiotic recombination, and amber nonsense mutations were located at different points on the map. The effect of the length of the labeling time on the specific radioactivity of ura 3 messenger RNA and on its repartition between poly(A)-RNA and RNA not containing poly(A) has been followed in nonsense mutants. Nonsense mutations reduce the messenger level without lowering its instantaneous rate of synthesis. The strength of the reduction depends on the position of the nonsense codon within the locus and concerns essentially the accumulation of polyadenylylated ura 3 mRNA.     

Hormonal control of prostatic thyrotropin-releasing hormone (TRH) testosterone modulates prostatic TRH concentrations

The presence of extremely high concentrations of authentic TRH in the rat prostate prompted us to examine whether prostatic TRH concentrations were under hormonal control. Both the immunoreactive TRH content per prostate and TRH immunoreactivity expressed per 100 mg prostatic protein were lower in animals 2 weeks after hypophysectomy than in sham-operated and calorically restricted weight-matched controls. Since many of the prostatic functions are testosterone dependent, we assessed the possibility that testosterone modulated prostatic TRH concentrations. We measured prostatic TRH concentrations in the following four groups of sexually mature male Sprague-Dawley rats: group I, sham operated; group II, castrated; group III, castrated animals with 5-mm Silastic testosterone implants; and group IV, castrated animals with 20-mm testosterone implants. Prostatic TRH concentrations in these four groups 2 weeks after surgery were 600.5 +/- 33.3, 65.1 +/- 22.6, 169.4 +/- 55.3, and 609.5 +/- 144.3 (+/-SE) ng/100 mg protein. There was good linear correlation between prostatic TRH concentrations and serum testosterone concentrations (r = 0.65; P less than 0.01). By subjecting the pooled prostatic extracts from each group to ion exchange chromatography on a SP-Sephadex C-25 column and measuring the proportion of immunoreactivity coeluting with authentic TRH, it was shown that the fall in prostatic TRH immunoreactivity after castration and hypophysectomy was indeed due to a loss of authentic TRH. We conclude that the prostatic TRH concentrations are under hormonal control and appear to be modulated by serum testosterone concentrations. This is the first demonstration of hormonal regulation of a neuropeptide in a mammalian extrahypothalamic site and suggests a physiological role for this neuropeptide at this site.     

Changes in Macromolecular Synthesis Associated with the Induction of Glutamine Synthetase in Embryonic Retina

The hydrocortisone-mediated induction of glutamine synthetase in the neural retina of chicken embryo in vitro is correlated with enhanced incorporation into protein of [(14)C]aspartic acid, an amino acid abundant in this enzyme. In the induced retina labeled with [(14)C]aspartic acid, a peak of radioactivity was detected in the region of the polysomal profile corresponding to polysomes comprising 12-14 ribosomes. In retinas labeled with [(3)H]uridine, an increased amount of radioactivity was also detected in the same polysomal region of the hydrocortisone-induced retina. If we assume a monocistronic messenger RNA for retinal glutamine synthetase, this region corresponds to the estimated size of the polysomes necessary for the translation of this enzyme. The evidence presented demonstrates a correlation between these changes in incorporation and the induction of glutamine synthetase.     

Gibberellic Acid Causes Increased Synthesis of RNA Which Contains Poly(A) in Barley Aleurone Tissue

Incubation of isolated barley aleurone layers with gibberellic acid for 16 hr caused a 50% increase in the synthesis of RNA that contains poly(A) sequences [poly(A)-RNA], but had no measurable effect on the syntheses of the major RNA species. The syntheses of both the poly(A) and the heteropolymeric fractions of the poly(A)-RNA were increased.The poly(A) sequences were separated into two classes by size, one containing an average of 250 nucleotides and the other about 70 nucleotides. The two classes occurred in a molar ratio of about 1:1. Gibberellic acid increased the syntheses of both sequences to the same extent.We interpret these results to mean that gibberellic acid increases specifically the synthesis of mRNA in this tissue.     

Molecular cloning of a vitamin D-dependent calcium-binding protein mRNA sequence from chick intestine.

We have constructed a recombinant cDNA library to facilitate study of the genomic actions of vitamin D3 and its hormonally active metabolite 1,25-dihydroxyvitamin D3 in initiation of the de novo biosynthesis of a 28,000-dalton vitamin D-dependent calcium binding protein (CaBP) present in chick intestine. The recombinant plasmids were prepared by the homopolymeric tailing and hybridization method using as a starting template poly(A)-enriched mRNA obtained from the intestinal mucosa of vitamin D3-replete (+D) chicks. Screening of 9,516 clones in this library was effected by using a comparative in situ colony hybridization technique with two [32P]cDNA probes; these probes were prepared from total poly(A)-RNA from chick intestinal mucosa of vitamin D-deficient (-D) chicks and a poly(A)-RNA specifically enriched for chick intestinal CaBP mRNA by immunoprecipitation of polysomes derived from vitamin D-replete (+D) chicks. We identified 26 clones that consistently displayed a significantly increased hybridization signal when comparing the -D vs. CaBP-enriched probe. Further evaluation of these clones by hybrid-selected translation showed the presence of CaBP-specific sequences. By "RNA gel" analysis of poly(A)-RNA, three independent mRNA species were found to hybridize to a CaBP clone; none of these RNA species were found in -D poly(A)-RNA. With this comparative colony hybridization procedure, we were able to identify CaBP-specific clones corresponding to a mRNA that is 0.1% of the total poly(A)-mRNA. The differential colony hybridization procedure using an enriched vs. a nonenriched probe should be of value in screening for other cDNA clones complementary to rare mRNA species.     

Testosterone stimulation of a rapidly labeled, low-molecular-weight RNA fraction in human hepatic erythroid cells in culture.

The addition of erythropoietin to cell cultures of erythroid cells of human fetal liver resulted in an increased incorporation of thymidine, adenine, and uridine into trichloroacetic acid-insoluble cell fractions and in an increased uptake of adenine and uridine into the cell. Although the effects of testosterone and erythropoietin on heme synthesis in these cells are known to be very similar, there was no effect of testosterone on the total incorporation of radioactive precursors into DNA or RNA. The RNA synthesized after short pulses of radioactive uridine, when analyzed on sucrose gradients containing 1% sodium dodecyl sulfate, consisted of a homogeneous peak sedimenting at 10 plus or minus 2 S, which is quite different from the heterogeneous, high-molecular-weight RNA synthesized under identical conditions in primary cultures of human fetal lung, kidney, or liver parenchymal cells. Addition of testosterone to liver erythroid cells in cultures for 5 hr followed by a 1-hr uridine pulse resulted in a 3-fold increase of RNA species with an average sedimentation coefficient of 14 plus or minus 3 S. The similarity with the sedimentation coefficient of the globin mRNA described in other systems and the high degree of specialization of the erythroid cells suggest that this RNA may be a stable intermediate involved in the synthesis of hemoglobin.     

A major species of mammalian messenger RNA lacking a polyadenylate segment.

Translation of total polysomal RNA from sarcoma 180 ascites cells in a wheat germ cell-free system produces two major polypeptides, A and B, with molecular weights of 50,000 and 45,000, respectively. Fractionation on Millipore filters or on oligo(dT)-cellulose leads to retention of the mRNA specific for protein A in the poly(A)-containing fraction and to accumulation of the B mRNA in the unadsorbed poly(A)-deficient fraction. The mRNA for B sediments at approximately 18 S; it is released as a 50S ribonucleorprotein upon EDTA treatment of polysomes. Its translation is particularly sensitive to an inhibitor present in the polysomal RNA. The poly(A)-deficient mRNA for the 45,000 dalton polypeptide is also present in mouse myeloma MPC-11 cells, where it seems to be localized in membrane-bound polysomes.     

The Role of Cytoplasmic Membranes in Controlling the Transport of Nuclear Messenger RNA and Initiation of Protein Synthesis

Three different classes of poly(A)-containing RNA have been identified in the yeast system. They have been characterized by their kinetics of labeling, by their localization in the nuclear, membranous, and soluble cytoplasmic fractions, and by their size. The evidence indicates that the membrane-bound poly(A)-containing RNA is a product of the nuclear poly(A)-containing RNA and is the precursor of the polysomal poly(A)-containing RNA. In addition, it has been demonstrated that transport of mRNA is coupled with protein synthesis.     

Translation of RNA that contains polyadenylate from yeast mitochondria in an Escherichia coli ribosomal system.

RNA that contains poly(A) [poly(A)-RNA] has been isolated from yeast mitochondria by poly(U) Sepharose-4B column chromatography. Pulse-labeled poly(A)-RNA shows 8-10 discrete peaks by acrylamide gel electrophoresis. The specific activity of mitochondrial poly(A)-RAN is six to eight times greater than that of mitochondrial rRNA after pulse labeling of protoplasts with [3H-]uridine. Ethidium bromide inhibits incorporation by over 90%. The total mitochondrial RNA preparation was contaminated with 5-15% cytoplasmic rRNA as determined by gel electrophoresis, but RNA exhaustion hybridization experiments indicated little or no cytoplasmic contamination of the mitochondrial poly(A)-RNA. The poly(A)-RNA stimulates [3H]leucine incorporation into protein in an E. coli cell-free system. A fraction of the labeled product is precipitated with antibody directed toward yeast cytochrome oxidase, but not with antibody directed toward bovine serum albumin. Sodium dodecyl sulfate gel electrophoresis of the immunoprecipitated material reveals labeled peptides having the mobility of the three larger cytochrome oxidase peptides, which are known to be translated by mitochondrial ribosomes.     

Two classes of translational control RNA: their role in the regulation of protein synthesis.

Two classes of translation control RNA (tcRNA) have been isolated from embryonic chick muscle. One of these classes, the tcRNA isolated from messenger ribonucleoprotein particles (mRNP-tcRNA), is effective in inhibiting the translation of mRNP-mRNA while having little if any effect on polysomal mRNA. The other class, polysome-tcRNA, has no effect on mRNP-mRNA while it stimulates the translation of polysomal mRNA. The mRNP tcRNA contains approximately 50 percent uridylate residues and forms small but stable hybrids with poly (A), while polysome-tcRNA contains fewer uridylate residues and is much less effective in forming a hybrid with poly (A). A proposed model concerning the role of these two classes of tcRNA in the regulation of protein synthesis is presented.     

Regulation of casein synthesis in the rabbit mammary gland. Titration of mRNA activity for casein under prolactin and progesterone treatments.

Total polysomal RNA or poly(A)-containing RNA isolated from membrane-bound polysomes of normal lactating rabbits directed the synthesis of casein in a reticulocyte lysate. Casein was identified by immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis of the immunoprecipitate. The poly(A)-containing RNA was heterogeneous with one major peak corresponding to a 12-S sedimentation coefficient as determined by polyacrylamide gel electrophoresis. Using the same procedure, mRNAs isolated from the non-secreting tissue of pseudopregnant rabbits were found not to contain the 12-S peak and were unable to direct the synthesis of casein in vitro. Prolactin injected into pseudopregnant rabbits induced the synthesis of proteins immunoprecipitable by anti-casein anti-serum and induced the simultaneous appearance of the 12-S mRNA. Progesterone injected with prolactin prevented the induction of casein synthesis and the appearance of mRNA for casein. A close relationship was established between the ability of the tissue to synthesize immunoprecipitable casein and the corresponding mRNA content of polysomes.     

Cellular and molecular aspects of thymus dependent antibody production in aged C3H/HeBr mice

An age related decline in anti-sheep erythrocyte antibody produced by lymphoid cells in splenic tissue of male C3H/HeBr mice was observed for both IgM and IgG production. The total number of lymphocytes per gram of splenic tissue was diminished, but there was no observed decrease in the spleen size with age. Analyses for enhanced suppressor cell activity as is found in tumor bearing mice of this strain revealed no enhanced suppressor activity in non-tumor bearing aged mice. Analyses of total cellular RNA from old and young mouse splenic lymphocytes revealed 8.5% less total RNA in aged mouse cells. Of the total RNA content, the percent of functional messenger RNA (poly(A)-RNA) declined 19.3% in the aged mouse cells. Isolation of the poly(A) tailed messenger RNA followed by cell-free translation of the message using a reticulocyte lysate system demonstrated that the poly(A)-RNA from young and aged mouse splenic lyphocytes directed protein synthesis to the same extent, indicating that although there was less poly(A)-RNA in the aged mouse cells, the activity of the poly(A)-RNA was relatively equivalent to that of young mouse cells. Translated proteins from poly(A)-RNA directed synthesis analyzed by SDS-polyacrylamide gel electrophoresis suggested a significant decline in immunoglobulin light and heavy chain message in the aged mouse lymphocytes of B cell enriched as well as unfractionated populations. This study suggested that age related declining humoral immunity in C3H/HeBr mice may have resulted from a defect in the B cell or helper T cell compartments, but was not the result of enhanced suppressor activity. This finding was substantiated by an observed decrease in poly(A)-RNA levels in aged mouse lymphoid cells as well as a suggested loss of intrinsic message for antibody synthesis.     

Sex differences in the metabolic effects of testosterone in sheep

Adiposity is regulated in a sexually divergent manner. This is partly due to sex steroids, but the differential effects of androgens in males and females are unclear. We investigated effects of testosterone on energy balance in castrated male (n = 6) and female sheep (n = 4), which received 3 × 200 mg testosterone implants for 2 wk or blank implants (controls). Temperature probes were implanted into retroperitoneal fat and skeletal muscle. Blood samples were taken to measure metabolites and insulin. In males, muscle and fat biopsies were collected to measure uncoupling protein (UCP) mRNA and phosphorylation of AMP-activated protein kinase and Akt. Testosterone did not change food intake in either sex. Temperature in muscle was higher in males than females, and testosterone reduced heat production in males only. In fat, however, temperature was higher in the castrate males compared with females, and there was no effect of testosterone treatment in either sex. Preprandial glucose levels were lower, but nonesterified fatty acids were higher in females compared with males, irrespective of testosterone. In males, the onset of feeding increased UCP1 and UCP3 mRNA levels in skeletal muscle, without an effect of testosterone. During feeding, testosterone reduced glucose levels in males only but did not alter the phosphorylation of AMP-activated protein kinase or Akt in muscle. Thus, testosterone maintains lower muscle and fat temperatures in males but not females. The mechanism underlying this sex-specific effect of testosterone is unknown but may be due to sexual differentiation of the brain centers controlling energy expenditure.