scholarly journals Developmentally Regulated Telomerase Activity Is Correlated with Chromosomal Healing during Chromatin Diminution inAscaris suum

1999 ◽  
Vol 19 (5) ◽  
pp. 3457-3465 ◽  
Author(s):  
Laurent Magnenat ◽  
Heinz Tobler ◽  
Fritz Müller
Keyword(s):  
Telomerase Activity ◽  
Embryonic Cell ◽  
Chromosomal Breakage ◽  
Chromatin Diminution ◽  
Developmentally Regulated ◽  
Cell Stage ◽  
Chromosome Healing ◽  
Cell Extracts ◽  
Dna Elimination

ABSTRACT Telomerase is the ribonucleoprotein complex responsible for the maintenance of the physical ends, or telomeres, of most eukaryotic chromosomes. In this study, telomerase activity has been identified in cell extracts from the nematode Ascaris suum. This parasitic nematode is particularly suited as a model system for the study of telomerase, because it shows the phenomenon of chromatin diminution, consisting of developmentally programmed chromosomal breakage, DNA elimination, and new telomere formation. In vitro, the A. suum telomerase is capable of efficiently recognizing and elongating nontelomeric primers with nematode-specific telomere repeats by using limited homology at the 3′ end of the DNA to anneal with the putative telomerase RNA template. The activity of this enzyme is developmentally regulated, and it correlates temporally with the phenomenon of chromatin diminution. It is up-regulated during the first two rounds of embryonic cell divisions, to reach a peak in 4-cell-stage embryos, when three presomatic blastomeres prepare for chromatin diminution. The activity remains high until the beginning of gastrulation, when the last of the presomatic cells undergoes chromatin diminution, and then constantly decreases during further development. In summary, our data strongly argue for a role of this enzyme in chromosome healing during the process of chromatin diminution.

scholarly journals A newly formed telomere in Ascaris suum does not exert a telomere position effect on a nearby gene.

1996 ◽  
Vol 16 (1) ◽  
pp. 130-134 ◽  
Author(s):  
Y J Huang ◽  
R Stoffel ◽  
H Tobler ◽  
F Mueller
Keyword(s):  
Dna Sequences ◽  
Developmental Stages ◽  
Ascaris Suum ◽  
Chromosomal Rearrangements ◽  
Position Effect ◽  
Start Codon ◽  
Chromosomal Breakage ◽  
Chromatin Diminution ◽  
Developmentally Regulated ◽  
Protein Encoding

During the process of chromatin diminution in Ascaris suum (formerly named Ascaris lumbricoides var. suum), developmentally regulated chromosomal fragmentation and new telomere addition occur within specific chromosomal breakage regions (CBRs). The DNA sequences flanking one of these CBRs (CBR-1) were analyzed, and two protein-encoding genes were found on either side. The noneliminated gene, agp-1, whose AUG start codon is located within approximately 2 kb of the boundary of CBR-1, encodes a putative GTP-binding protein which is structurally related to eukaryotic and prokaryotic elongation factors. Northern (RNA) blot analyses revealed that transcripts of this gene are present at all developmental stages, suggesting that the massive chromosomal rearrangements associated with the process of chromatin diminution have no influence on agp-1 expression. This demonstrates that addition of new telomeres in CBR-1 does not result in a telomeric position effect, a phenomenon previously described in Saccharomyces cerevisiae.

scholarly journals Proteolysis of the major yolk glycoproteins is regulated by acidification of the yolk platelets in sea urchin embryos

1992 ◽  
Vol 117 (6) ◽  
pp. 1211-1221 ◽  
Author(s):  
SK Mallya ◽  
JS Partin ◽  
MC Valdizan ◽  
WJ Lennarz
Keyword(s):  
Early Development ◽  
Sea Urchin ◽  
Ph Value ◽  
Total Protein Content ◽  
Developmentally Regulated ◽  
Cell Stage ◽  
Ph Optimum ◽  
Sea Urchin Embryos

The precise function of the yolk platelets of sea urchin embryos during early development is unknown. We have shown previously that the chemical composition of the yolk platelets remains unchanged in terms of phospholipid, triglyceride, hexose, sialic acid, RNA, and total protein content after fertilization and early development. However, the platelet is not entirely static because the major 160-kD yolk glycoprotein YP-160 undergoes limited, step-wise proteolytic cleavage during early development. Based on previous studies by us and others, it has been postulated that yolk platelets become acidified during development, leading to the activation of a cathepsin B-like yolk proteinase that is believed to be responsible for the degradation of the major yolk glycoprotein. To investigate this possibility, we studied the effect of addition of chloroquine, which prevents acidification of lysosomes. Consistent with the postulated requirement for acidification, it was found that chloroquine blocked YP-160 breakdown but had no effect on embryonic development. To directly test the possibility that acidification of the yolk platelets over the course of development temporally correlated with YP-160 proteolysis, we added 3-(2,4-dinitroanilo)-3-amino-N-methyldipropylamine (DAMP) to eggs or embryos. This compound localizes to acidic organelles and can be detected in these organelles by EM. The results of these studies revealed that yolk platelets did, in fact, become transiently acidified during development. This acidification occurred at the same time as yolk protein proteolysis, i.e., at 6 h after fertilization (64-cell stage) in Strongylocentrotus purpuratus and at 48 h after fertilization (late gastrula) in L. pictus. Furthermore, the pH value at the point of maximal acidification of the yolk platelets in vivo was equal to the pH optimum of the enzyme measured in vitro, indicating that this acidification is sufficient to activate the enzyme. For both S. purpuratus and Lytechinus pictus, the observed decrease in the pH was approximately 0.8 U, from 7.0 to 6.2. The trypsin inhibitor benzamidine was found to inhibit the yolk proteinase in vivo. By virtue of the fact that this inhibitor was reversible we established that the activity of the yolk proteinase is developmentally regulated even though the enzyme is present throughout the course of development. These findings indicate that acidification of yolk platelets is a developmentally regulated process that is a prerequisite to initiation of the catabolism of the major yolk glycoprotein.

scholarly journals Identification of Kluyveromyces lactisTelomerase: Discontinuous Synthesis along the 30-Nucleotide-Long Templating Domain

1998 ◽  
Vol 18 (9) ◽  
pp. 4961-4970 ◽  
Author(s):  
Tracy Boswell Fulton ◽  
Elizabeth H. Blackburn
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Kluyveromyces Lactis ◽  
Telomerase Activity ◽  
Reaction Products ◽  
Telomeric Dna ◽  
Whole Cell ◽  
Cell Extracts ◽  
Dna Template ◽  
Insight Into

ABSTRACT Telomeres in the budding yeast Kluyveromyces lactisconsist of perfectly repeated 25-bp units, unlike the imprecise repeats at Saccharomyces cerevisiae telomeres and the short (6- to 8-bp) telomeric repeats found in many other eukaryotes. Telomeric DNA is synthesized by the ribonucleoprotein telomerase, which uses a portion of its RNA moiety as a template. K. lactistelomerase RNA, encoded by the TER1 gene, is ∼1.3 kb long and contains a 30-nucleotide templating domain, the largest ever examined. To examine the mechanism of polymerization by this enzyme, we identified and analyzed telomerase activity from K. lactiswhole-cell extracts. In this study, we exploited the length of the template and the precision of copying by K. lactistelomerase to examine primer elongation within one round of repeat synthesis. Under all in vitro conditions tested, K. lactistelomerase catalyzed only one round of repeat synthesis and remained bound to reaction products. We demonstrate that K. lactistelomerase polymerizes along the template in a discontinuous manner and stalls at two specific regions in the template. Increasing the amount of primer DNA-template RNA complementarity results in stalling, suggesting that the RNA-DNA hybrid is not unpaired during elongation in vitro and that lengthy duplexes hinder polymerization through particular regions of the template. We suggest that these observations provide an insight into the mechanism of telomerase and its regulation.

219 EXPRESSION PATTERN OF THE SUB-CORTICAL MATERNAL COMPLEX IN OVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 258 ◽  
Author(s):  
D. Bebbere ◽  
L. Bogliolo ◽  
F. Ariu ◽  
O. Murrone ◽  
A. Strina ◽  
...  
Keyword(s):  
Expression Pattern ◽  
Protein Complex ◽  
Mammalian Species ◽  
Expression Patterns ◽  
Embryonic Cell ◽  
Specific Pattern ◽  
Pcr Analysis ◽  
Cell Stage ◽  
Oocyte Growth

The sub-cortical maternal complex (SCMC) is a multi-protein complex located in the sub-cortex of the oocyte. In mouse, it assembles during oocyte growth and is essential for zygotes to progress beyond the first embryonic cell divisions (Li et al., 2008). At least 4 proteins contribute to the complex: oocyte expressed protein (OOEP), maternal antigen that embryo requires (MATER), transducin-like enhancer of split 6 (TLE6), and ES cell associated transcript 1 (ECAT1), all encoded by maternal effect genes. In mouse, the relative transcripts are degraded during meiotic maturation and ovulation, whereas the SCMC proteins persist in the early embryo. Whereas MATER expression has been studied in several species, the existence of the genes encoding the other components has been assessed in few mammalian species and their pattern of expression during pre-implantation development has been analysed only in mouse (Li et al. 2008 Dev. Cell 15, 416–425). In a previous work (Bebbere et al. 2008 Reprod. Fertil. Dev. 20, 908–915), we assessed MATER existence and pattern of expression in the ovine species. The aim of the present work was to assess the existence of OOEP, TLE6, and FILIA in the ovine species and to analyse the expression pattern of the 4 genes in the oocytes and during pre-implantation embryo development. Total RNA was isolated and reverse transcribed from pools of immature (GV) and in vitro matured (IVM) metaphase II (MII) oocytes, from in vitro fertilized and cultured (IVFC) embryos at the 2-, 4-, 8-, and 16-cell stage and from blastocysts. Three pools of 10 oocyte/embryos were analysed for each class. Primers were designed on the basis of the sequences conserved among orthologs and amplify intron-spanning regions. The PCR products were sequenced, and the alignment, performed with BLASTn, confirmed the homology with the orthologous genes present in public databases. Real-time PCR analysis revealed that all 4 transcripts are present at its highest level in the GV oocyte but decrease during embryo pre-implantation development with a gene-specific pattern. Conversely to the pattern of expression observed in mouse, all 4 transcripts persisted until the 8-cell stage embryo, disappearing only at the 16-cell stage. No transcripts were detected at the blastocyst stage. This study confirms the existence of transcripts related to SCMC also in the ovine species, but highlights species-specific patterns of expression in the 2 species, possibly related to the different time of activation of the embryo genome in mouse and in sheep. The observed expression patterns suggest an involvement of the protein complex in oocyte maturation and in the very first phases of life, possibly in the transition from the maternal to embryonic program of development.

Exogenous platelet-activating factor stimulates cell proliferation in mouse pre-implantation embryos prior to the fourth cell cycle and shows isoform-specific stimulatory effects

Zygote ◽  
2001 ◽  
Vol 9 (3) ◽  
pp. 261-268 ◽  
Author(s):  
Neil R. Stoddart ◽  
William E. Roudebush ◽  
Steven D. Fleming
Keyword(s):  
Cell Proliferation ◽  
Biological Activities ◽  
Platelet Activating Factor ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Embryonic Cell ◽  
Cell Stage ◽  
Cell Numbers ◽  
Paf Receptor

Mouse embryos secrete molecules homologous to platelet-activating factor (PAF), termed embryo-derived PAF (EPAF), which act in an autocrine/paracrine fashion to stimulate embryonic development in vitro. Mouse EPAF is thought to consist predominantly of hexadecyl (C16) and octadecyl (C18) PAF-like components. Mouse pre-implantation embryos cultured with exogenous PAF from the early cleavage stages exhibit increased blastocyst cell numbers and rates of mitosis around the 8-cell stage. We investigated whether exogenous PAF could specifically stimulate embryonic cell proliferation prior to the blastocyst stage in the mouse and also compared the biological activities of the C16 and C18 PAF isoforms as follows. Embryos were cultured for either 24 h or 120 h from the 2-cell stage and their total cell numbers were determined or their development assessed in terms of their incidence of successful zona-hatching respectively. In each case, embryos were cultured in unsupplemented medium or in medium supplemented with either C16 or C18 PAF (0.5 μM). Compared with controls, culture with C16 PAF produced a significant stimulation of mean total per number per embryo and a significant increase in the incidence of successful zona-hatching, whilst culture with C18 PAF had no significant effect. We then cultured 1-cell zygotes for 48 h in unsupplemented medium or medium supplemented with either an equimolar mixture of C16 and C18 PF or with either C16 or C18 PF alone (each at 0.2 μM). Embryos were also scored for cell number at 4 h and 30 h of culture. Although no significant effect on mean cell number per embryo was seen following 4 h or 30 h of culture with a mixed C16/C18 PAF preparation, culture for 48 h with a mixed C16/C18 PAF preparation or with C16 PAF alone produced a significant increase in mean cell number per embryo compared with controls - an effect that is likely to be receptor-mediated, since culture with an equivalent concentration of C18 PAF had no significant effect compared with controls. We have demonstrated that mouse zygotes/embryos can respond in a specific manner to exogenous hexadecyl PAF in terms of increased rates of cell proliferation prior to cavitation, and must be capable of doing so at some time between the first and third, and also between the second and fourth, cell cycles. Such embryos presumably express one or more classes of functional PAF-receptor molecule during this period (i.e. as early as during the 1-, 2- or 4-cell stages). We have also demonstrated that embryonic response to exogenous PAF is significantly isoform-specific, which may reflect differences between the two isoforms either in affinity for binding to putative embyronic PAF-receptor molecules or in their ability to elicit a stimulatory response following binding. This observation calls into question the use of preparations containing a mixture of hexadecyl and octadecyl PAF isoforms, particularly in dose-response studies, in the mouse.

91 INHIBITION OF TELOMERASE REVERSE TRANSCRIPTASE (TERT) INDUCES EMBRYO ARREST IN BOVINE PRE-IMPLANTATION EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 146
Author(s):  
P. Madan ◽  
W. A. King ◽  
D. H. Betts
Keyword(s):  
Reverse Transcriptase ◽  
Critical Role ◽  
Germinal Vesicle ◽  
Telomerase Activity ◽  
Blastocyst Stage ◽  
Telomerase Reverse Transcriptase ◽  
Dapi Staining ◽  
Cell Stage ◽  
Telomerase Inhibitor

Previous studies from our laboratory have detected telomerase activity in embryos from all stages of early bovine development. However, the regulation of the telomerase subunits remains poorly understood. The objective of this study was to characterize the expression and function of the bovine telomerase reverse transcriptase (bTERT) subunit during bovine pre-implantation embryogenesis. Using RT-PCR and immunofluorescence staining procedures (n = 20 embryos at timed stages of development; r = 3), we demonstrate that mRNA transcripts and protein for bTERT were detected in pre-implantation bovine embryos from 1-cell to the blastocyst stage. The specificity of bTERT PCR products was confirmed by sequencing and exhibited 94% sequence homology to the human hTERT cDNA sequence. In immature oocytes, the bTERT protein was localized within the germinal vesicle and after 18 to 20 h of in vitro maturation, bTERT was observed as doublet foci co-localized with the condensed metaphase II meiotic stage chromosomes. Post-fertilization, the expression of bTERT was observed within the pronuclei. Through the initial cleavage stages, the expression of bTERT was variable, with some blastomeres showing punctate staining, whereas others exhibited a more-uniform staining pattern within the cytoplasm. By the 8- to 16-cell stage, the embryos demonstrated a peri-nuclear presence of bTERT. However, while in morulae and blastocysts, bTERT was localized to the nuclei as demonstrated by co-localization with 4′,6-diamidino-2-phenylindole (DAPI) staining. Treatment of zygotes with Telomerase Inhibitor III (telomere mimic that inhibits telomerase activity; Calbiochem, San Diego, CA) significantly induced embryos to permanently arrest in a senescence-like state at the 2- to 4-cell cleavage stage compared with nontreated controls. These arrested embryos showed abnormal nuclear phenotypes (elongated chromosomes, anaphase bridging) and phosphorylated histone γ-H2A.X foci suggesting a critical role(s) of TERT in chromosomal segregation and telomere integrity in early embryos. The translocation of bTERT protein from the cytoplasm to the nucleus in morulae supports the known telomere elongation event, whereas low levels of TERT at the 2- to 4-cell stage are associated with the previously documented state of permanent embryo arrest. Future studies would be directed toward understanding the relationship between TERT expression, telomere dysfunction, and permanent embryo arrest. Operating grants from the Canadian Institutes of Health Research (CIHR) to D.H.B. and W.A.K. supported this research.

111 CHARACTERIZATION OF BOVINE TELOMERASE REVERSE TRANSCRIPTASE DURING PREIMPLANTATION EMBRYOGENESIS

2008 ◽  
Vol 20 (1) ◽  
pp. 136
Author(s):  
P. Madan ◽  
W. A. King ◽  
D. H. Betts
Keyword(s):  
Reverse Transcriptase ◽  
Germinal Vesicle ◽  
Telomerase Activity ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Telomerase Reverse Transcriptase ◽  
Telomeric Dna ◽  
Cell Stage ◽  
Highly Active

Telomerase is a specialized reverse transcriptase that extends telomeric DNA of eukaryotic chromosomes and is composed of 2 essential subunits, the telomerase RNA component (TERC) and the telomerase reverse transcriptase (TERT). Together, this ribonucleoprotein complex is normally highly active in germ cells and stem/progenitor cells but not in normal somatic cells. In humans, this regulation of telomerase activity is primarily coordinated at the level of transcription of the TERT gene, whereas TERC is virtually ubiquitously expressed. Previous studies from our laboratory have detected telomerase activity in embryos from all stages of early bovine development. However, the regulation of the telomerase subunits remains poorly understood. Therefore, the objective of our study was to characterize the expression of bovine TERT (bTERT) during bovine preimplantation embryogenesis. UsingRT-PCR and immunofluorescence staining procedures (n = 20 embryos at timed stages of development; r = 3), we demonstrate that mRNA transcripts and protein for bTERT were detected in preimplantation bovine embryos from 1-cell to the blastocyst stage. The specificity of bTERT PCR products was confirmed by sequencing and demonstrated 94% sequence homology to the human hTERT cDNA sequence. In the immature oocyte, bTERT protein was localized within the germinal vesicle, and after 18–20 h of in vitro maturation, bTERT was observed as doublet foci co-localized with the condensed metaphase II meiotic stage chromosomes. Post-fertilization, the expression of bTERT was observed within the pronuclei. Through the initial cleavage stages, the expression of bTERT was variable, with some blastomeres showing punctuate staining and others exhibiting a uniform staining pattern. By the 8-16 cell stage, the embryos demonstrated a peri-nuclear presence of bTERT. However, in morula and blastocysts, bTERT was localized to the nuclei as demonstrated by co-localization with 42,6-diamidino-2-phenylindole staining. Based on this pattern of expression, it is tempting to speculate that bTERT could be playing an important role in regulating telomerase activity during early embryo development. The translocation of bTERT protein from the cytoplasm to the nucleus in morulae supports the telomere elongation event that is known to occur in mammalian embryos during the transition from the morula to blastocyst stages.

Chromatin diminution in the copepod Mesocyclops edax: elimination of both highly repetitive and nonhighly repetitive DNA

Genome ◽  
2013 ◽  
Vol 56 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Christian McKinnon ◽  
Guy Drouin
Keyword(s):  
Repetitive Dna ◽  
Tandem Repeats ◽  
Repetitive Sequences ◽  
Embryonic Cells ◽  
Chromatin Diminution ◽  
Developmentally Regulated ◽  
Dna Elimination ◽  
Eukaryotic Species ◽  
Cyclops Kolensis ◽  
Mesocyclops Edax

Chromatin diminution, a developmentally regulated process of DNA elimination, is found in numerous eukaryotic species. In the copepod Mesocyclops edax, some 90% of its genomic DNA is eliminated during the differentiation of embryonic cells into somatic cells. Previous studies have shown that the eliminated DNA contains highly repetitive sequences. Here, we sequenced DNA fragments from pre- and postdiminution cells to determine whether nonhighly repetitive sequences are also eliminated during the process of chromatin diminution. Comparative analyses of these sequences, as well as the sequences eliminated from the genome of the copepod Cyclops kolensis, show that they all share similar abundances of tandem repeats, dispersed repeats, transposable elements, and various coding and noncoding sequences. This suggests that, in the chromatin diminution observed in M. edax, both highly repetitive and nonhighly repetitive sequences are eliminated and that there is no bias in the type of nonhighly repetitive DNA being eliminated.

Effect of embryonic cell cycle of nuclear donor embryos on the efficiency of nuclear transfer in Japanese black cattle

Zygote ◽  
2007 ◽  
Vol 15 (2) ◽  
pp. 165-171
Author(s):  
M. Kishi ◽  
R. Takakura ◽  
Y. Nagao ◽  
K. Saeki ◽  
Y. Takahashi
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Embryonic Cell ◽  
Embryonic Cells ◽  
Japanese Black Cattle ◽  
Cell Stage ◽  
Cell Cycle Stage

SummaryIn the present study, the development in vitro and in vivo of nuclear transfer (NT) embryos reconstructed with embryonic cells (blastomeres) at the 32- to 63-cell (sixth cell cycle) and 64- to 127-cell (seventh cell cycle) stages was investigated to determine the optimum range of embryonic cell cycles for yielding the highest number of identical calves in Japanese black cattle. Rates of development to the blastocyst stage (overall efficiency) were higher in the sixth cell-cycle stage (45%) than in the seventh cell-cycle stage (12%). After the transfer of the blastocysts reconstructed with blastomeres of the sixth and seventh cell cycle-stage embryos to recipient heifers, there were no differences in the pregnancy (14/35: 40% versus 3/13: 23%, respectively) or calving rates (11/39: 28% versus 3/13: 23%, respectively). These results indicate that the highest number of identical calves would be obtained by using sixth cell cycle (32- to 63-cell)-stage embryos as nuclear donors.

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