Changes in protein kinase activity in rat testes during development.
Ontology highlight
ABSTRACT:
SUBMITTER: Bernard EA
PROVIDER: S-EPMC1164622 | biostudies-other | 1977 Feb
REPOSITORIES: biostudies-other
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Project description:A-kinase anchoring proteins (AKAPs) bind the regulatory subunits of protein kinase A (PKA) and localize the holoenzyme to discrete signaling microdomains in multiple subcellular compartments. Despite emerging evidence for a nuclear pool of PKA that rapidly responds to activation of the PKA signaling cascade, only a few AKAPs have been identified that localize to the nucleus. Here we show a PKA-binding domain in the amino terminus of Chd8, and demonstrate subcellular colocalization of Chd8 with RII. RII overlay and immunoprecipitation assays demonstrate binding between Chd8-S and RII?. Binding is abrogated upon dephosphorylation of RII?. By immunofluorescence, we identified nuclear and perinuclear pools of Chd8 in HeLa cells and rat neonatal cardiomyocytes. We also show high levels of Chd8 mRNA in RNA extracted from post-natal rat hearts. These data add Chd8 to the short list of known nuclear AKAPs, and implicate a function for Chd8 in post-natal rat cardiac development.
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Project description:1. The activity of thymidine kinase in rat liver supernatant decreased with development to a value in the adult that was 1% of that in the 17-day foetus. 2. The foetal enzyme was more stable than the adult to gel filtration on Sephadex G-25 at 0 degrees . 3. The greater stability of the foetal enzyme to incubation at 45 degrees was attributable to the presence of higher concentrations of nucleotides in foetal liver supernatant. 4. The K(m) values for foetal and adult enzymes were approx. 2.5mum- and 2.1mum-thymidine respectively. 5. The foetal enzyme was more sensitive to inhibition by thymidine triphosphate. 6. The decline in enzyme activity during the neonatal period was correlated with a shift in the enzyme properties from the foetal to the adult type, and may reflect the decrease in the proportion of haemopoietic tissue in the liver.
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Project description:BACKGROUND: Fluoxetine, a selective serotonin reuptake inhibitor, is approved for treatment of childhood depression. In rats, fluoxetine influences neuronal development, but it is unclear whether it also influences glia development. S100B is a glia-derived calcium-binding protein, which may influence the development of serotonergic fibers and, vice versa, serotonin may influence the expression of S100B. OBJECTIVES: The purpose of this study was to investigate whether fluoxetine treatment influences the expression of S100B during postnatal development, and whether potential changes are regionally dependent upon the time frame of drug administration. METHODS: S100B gene expression and S100B protein expression in three different brain regions (frontal cortex, hippocampus, and striatum) were studied by real-time polymerase chain reaction (PCR) and immunohistochemistry, respectively. First, a short-term effect, 24 hours after a 14 day fluoxetine treatment (5 mg/kg/bw s.c.) of rats either from postnatal day (PD) 1 to 15, 21 to 35, or 50 to 64, was investigated. Then, the same treatment was used to analyze S100B gene and protein levels at PD 90 (long-term effect). RESULTS: At PD 90, a significant increase of gene and protein expression was observed in all regions if rats were treated during PDs 21-35, whereas treatment during other periods had no long-term effects. A short-term effect 24 hours after fluoxetine treatment was found for almost all development stages and regions, demonstrated by a significant increase of S100B. CONCLUSIONS: These results support recent research indicating a highly drug-sensitive period (i.e., periadolescence) of rat brain development. Therefore, further clinical studies should be performed to clarify whether such a sensitive period also exists in children.
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Project description:During embryonic development, protein kinase A (PKA) plays a key role in cell fate specification by antagonizing the Hedgehog (Hh) signaling pathway. However, the mechanism by which PKA activity is regulated remains unknown. Here we show that the Misty somites (Mys) protein regulates the level of PKA activity during embryonic development in zebrafish. We isolate PKA regulatory type Ialpha subunit (Prkar1a) as a protein interacting with Mys by pulldown assay in HEK293 cells followed by mass spectrometry analysis. We show an interaction between endogenous Mys and Prkar1a in the zebrafish embryo. Mys binds to Prkar1a in its C terminus region, termed PRB domain, and activates PKA in vitro. Conversely, knockdown of Mys in zebrafish embryos results in reduction in PKA activity. We also show that knockdown of Mys induces ectopic activation of Hh target genes in the eyes, neural tube, and somites downstream of Smoothened, a protein essential for transduction of Hh signaling activity. The altered patterning of gene expression is rescued by activation of PKA. Together, our results reveal a molecular mechanism of regulation of PKA activity that is dependent on a protein-protein interaction and demonstrate that PKA activity regulated by Mys is indispensable for negative regulation of the Hh signaling pathway in Hh-responsive cells.
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Project description:1. Changes in the activities of ATP citrate lyase, ;malic' enzyme, glucose 6-phosphate dehydrogenase, pyruvate kinase and fructose 1,6-diphosphatase, and in the ability to incorporate [1-(14)C]acetate into lipid have been measured in the livers of developing rats between late foetal life and maturity. 2. In male rats the activities of those systems directly or indirectly concerned in lipogenesis (acetate incorporation into lipid, ATP citrate lyase and glucose 6-phosphate dehydrogenase) fall after birth and are maintained at a low value until weaning. After weaning these activities rise to a maximum between 30 and 40 days and then decline, reaching adult values at about 60 days. ;Malic' enzyme activity follows a similar course, except that none could be detected in the foetal liver. Pyruvate kinase activity is lower in foetal than in adult livers and rises to slightly higher than the adult value in the post-weaning period. Fructose 1,6-diphosphatase activity rises from a very low foetal value to reach a maximum at about 10 days but falls rapidly after weaning to reach adult values at about 30 days. 3. Weaning rats on to a high-fat diet caused the low activities of acetate incorporation, ATP citrate lyase, glucose 6-phosphate dehydrogenase and pyruvate kinase, characteristic of the suckling period, to persist. ;Malic' enzyme and fructose 1,6-diphosphatase activities were not altered appreciably. 4. No differences could be detected in hepatic enzyme activities between males and females up to 35 days, but after this time female rats gave higher values for acetate incorporation, glucose 6-phosphate dehydrogenase activity and ;malic' enzyme activity. 5. The results are discussed in relation to changes in alimentation and hormonal influences.
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Project description:1. Lipogenesis, as measured by the incorporation of (14)C-labelled glucose or acetate into fatty acids in liver slices, is high in foetal and adult rat liver but is low in the liver of the suckling rat, especially with glucose as substrate. 2. The rate of synthesis of non-saponifiable lipids from glucose is about 15 times as great in the liver of the 18-day foetus as in adult liver. Activity in the newborn is negligible. 3. Glucose incorporation into fat is strongly concentration-dependent in liver slices from the adult and 2-week-old rat, but less markedly so in liver slices from the foetus. 4. Changes in the activity of hepatic citrate-cleavage enzyme (ATP-citrate lyase) occur in parallel with the changes in the extent of fatty acid formation, supporting the participation of this enzyme in lipogenesis. However, NADP-malate dehydrogenase, a potential source of reduced nucleotide coenzyme for lipogenesis in the adult, could not be detected in foetal rat liver.
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Project description:Sertoli cells (Sc) are unique somatic cells of testis that are the target of both FSH and testosterone (T) and regulate spermatogenesis. Although Sc of neonatal rat testes are exposed to high levels of FSH and T, robust differentiation of spermatogonial cells becomes conspicuous only after 11-days of postnatal age. We have demonstrated earlier that a developmental switch in terms of hormonal responsiveness occurs in rat Sc at around 12 days of postnatal age during the rapid transition of spermatogonia A to B. Therefore, such "functional maturation" of Sc, during pubertal development becomes prerequisite for the onset of spermatogenesis. However, a conspicuous difference in robust hormone (both T and FSH) induced gene expression during the different phases of Sc maturation restricts our understanding about molecular events necessary for the spermatogenic onset and maintenance. Here, using microarray technology, we for the first time have compared the differential transcriptional profile of Sc isolated and cultured from immature (5 days old), maturing (12 days old) and mature (60 days old) rat testes. Our data revealed that immature Sc express genes involved in cellular growth, metabolism, chemokines, cell division, MAPK and Wnt pathways, while mature Sc are more specialized expressing genes involved in glucose metabolism, phagocytosis, insulin signaling and cytoskeleton structuring. Taken together, this differential transcriptome data provide an important resource to reveal the molecular network of Sc maturation which is necessary to govern male germ cell differentiation, hence, will improve our current understanding of the etiology of some forms of idiopathic male infertility.
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Project description:BACKGROUND: Androgen-dependent proteins (lipocalins) circulate in blood of male rats and mice and, being small (~ 18 kDa), pass freely into glomerular filtrate. Some are salvaged in proximal nephrons but some escape in urine. Several organic molecules can bind to these proteins causing, where salvage occurs, nephropathy including malignancy in renal cortex. In urine, both free lipocalins and ligands contribute to an increasingly-recognised vital biological role in social communication between adults, especially in the dark where reliance is on smell and taste. Crystal structure of the first-characterised lipocalin of male rats, ?2u-globulin, has been determined and peptide sequences for others are available, but no study of occurrence during early puberty has been made. We have followed temporal occurrence in urine of juveniles (n?=?3) for non-invasive pilot study by high resolution gradient mini-gel electrophoresis, tryptic digest of excised protein bands, and LC-MS/MS of digest to identify peptide fragments and assign to specific lipocalins. Study objective refers directly to external availability for social communication but also indirectly to indicate kinetics of circulating lipocalins to which some xenobiotics may bind and constitute determinants of renal disease. RESULTS: Mini-gels revealed greater lipocalin complexity than hitherto recognised, possibly reflecting post-translational modifications. Earliest patterns comprised rat urinary protein 1, already evident in Sprague-Dawley and Wistar strains at 36 and 52 days, respectively. By 44 and 57 days major rat protein (?2u-globulin) occurred as the progressively more dominant protein, though as two forms with different electrophoretic mobility, characterised by seven peptide sequences. No significant change in urinary testosterone had occurred in Wistars when major rat protein became evident, but testosterone surged by 107 days concomitant with the marked abundance of excreted lipocalins. CONCLUSIONS: Qualitative temporal changes in the composition of excreted lipocalins early in puberty, and apparent increase in major urinary protein as two resolvable forms, should catalyse systematic non-invasive study of urinary lipocalin and testosterone dynamics from early age, to illuminate this aspect of laboratory rodent social physiology. It could also define the potential temporal onset of nephrotoxic ligand risk, applicable to young animals used as toxicological models.
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