402. Cryopreservation of oocytes and follicular cells of the cane toad Bufo Marinus

2008 ◽  
Vol 20 (9) ◽  
pp. 82
Author(s):  
K. H. Wooi ◽  
M. J. Mahony ◽  
J. M. Shaw ◽  
J. Clulow
Keyword(s):  
Developmental Stages ◽  
Slow Cool ◽  
Ovarian Follicles ◽  
Stage I ◽  
Follicle Cells ◽  
Follicular Cells ◽  
Slow Cooling ◽  
Technical Problems

Amphibians are currently the most threatened of all vertebrate groups with more than 30% of all known species in decline, facing extinction or recently extinct. Cryobanking of amphibian germ cells and reproductive tissues could be used to manage threatened species and provide insurance against extinction. However, cryopreservation of fully developed amphibian oocytes and whole embryos has not been achieved due to technical problems freezing such large cellular structures. As an alternative approach, we investigated the feasibility of developing protocols for the slow-cool freezing, storage and retrieval of developmentally competent amphibian ovarian follicles containing Stage I and II oocytes which are much smaller in size than later developmental stages. Ovarian follicles from euthanased Cane Toads were incubated in cryodiluents containing either glycerol or DMSO to assess cryoprotectant toxicity and response to slow cooling freezing protocols. The fluorescent live cell stain SYBR 14 and its counter stain propidium iodide was used to score the proportion of viable follicle cells before and after cryopreservation. Cryoprotectant type, concentration and exposure time all had significant effects (P < 0.05) on the viability of follicle cells, with significant interactions between these variables. Overall, glycerol was less toxic to follicle cells than DMSO. At higher concentrations, glycerol exerted high osmotic stress on oocytes, and there was evidence that DMSO triggered apoptosis in oocytes. The most effective cryopreservation protocol for stage I and II oocyte follicles resulted in a post-thaw recovery of a mean 70% of viable follicular cells. This protocol involved cryopreservation in 15% v/v glycerol, inclusion of seeding and temperature holding periods during cryopreservation, coupled with rapid thawing in a 30°C water bath. The successful cryopreservation of intact follicles in this study indicates the potential to recover functional ovarian tissues post cryopreservation for continuation of amphibian oogenesis in vitro or in vivo.

Ultrastructural analysis of Drosophila ovarian follicles differing in yolk polypeptide (yps) composition

Development ◽  
1993 ◽  
Vol 117 (1) ◽  
pp. 319-328
Author(s):  
F. Giorgi ◽  
P. Lucchesi ◽  
A. Morelli ◽  
M. Bownes
Keyword(s):  
Golgi Apparatus ◽  
Juvenile Hormone ◽  
Developmental Stages ◽  
Ovarian Follicles ◽  
Endocytic Pathway ◽  
Intermediate Cell ◽  
Wild Type ◽  
Vesicular Traffic

Drosophila ovarian follicles were examined ultrastructurally to study the vesicular traffic in the cortical ooplasm. The endocytic pathway leading to the production of yolk spheres was visualized following in vivo or in vitro exposure to peroxidase. The Golgi apparatus and the yolk spheres of wild-type ovarian follicles were preferentially labelled by fixation with osmium zinc iodide (OZI). Labelling of wild-type ovarian follicles was compared to that of several mutant follicles--L186/Basc, fs(2)A17 and ap4--which are defective in vitellogenesis. In these mutants, the Golgi apparatus and the vesicles nearby were either scantly labelled or not labelled at all. In oocytes from flies homozygous for the gene fs(1)1163, the Golgi apparatus was labelled as in the controls, but no yolk spheres appeared to be labelled with OZI at any of the developmental stages. In several Drosophila strains, the pattern of OZI label in the cortical ooplasm was seen to vary in relation to the number of yp structural genes. In starved Drosophila females, OZI labelling of the cortical ooplasm appeared restricted to the Golgi apparatus and to an extended tubular network. A similar labelling pattern was also detected in in vitro cultured vitellogenic follicles. Refeeding, topical application of juvenile hormone analogue to starved females or hormone addition to the culture medium, all caused the yolk spheres to become labelled with OZI and to incorporate peroxidase. These observations prove that impairing endocytic uptake by either mutation or lack of juvenile hormone prevents fusion of coated vesicles and tubules with the yolk spheres and leads them instead to form an intermediate cell compartment with Golgi-derived vesicles.

scholarly journals Genome-Wide Transcriptomic Identification and Functional Insight of Lily WRKY Genes Responding to Botrytis Fungal Disease

Plants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 776
Author(s):  
Shipra Kumari ◽  
Bashistha Kumar Kanth ◽  
Ju young Ahn ◽  
Jong Hwa Kim ◽  
Geung-Joo Lee
Keyword(s):  
Abiotic Stress ◽  
Biotic Stress ◽  
Developmental Stages ◽  
Expression Patterns ◽  
Lilium Longiflorum ◽  
Biotic And Abiotic Stress ◽  
Biotic Stresses ◽  
Genome Wide

Genome-wide transcriptome analysis using RNA-Seq of Lilium longiflorum revealed valuable genes responding to biotic stresses. WRKY transcription factors are regulatory proteins playing essential roles in defense processes under environmental stresses, causing considerable losses in flower quality and production. Thirty-eight WRKY genes were identified from the transcriptomic profile from lily genotypes, exhibiting leaf blight caused by Botrytis elliptica. Lily WRKYs have a highly conserved motif, WRKYGQK, with a common variant, WRKYGKK. Phylogeny of LlWRKYs with homologous genes from other representative plant species classified them into three groups- I, II, and III consisting of seven, 22, and nine genes, respectively. Base on functional annotation, 22 LlWRKY genes were associated with biotic stress, nine with abiotic stress, and seven with others. Sixteen unique LlWRKY were studied to investigate responses to stress conditions using gene expression under biotic and abiotic stress treatments. Five genes—LlWRKY3, LlWRKY4, LlWRKY5, LlWRKY10, and LlWRKY12—were substantially upregulated, proving to be biotic stress-responsive genes in vivo and in vitro conditions. Moreover, the expression patterns of LlWRKY genes varied in response to drought, heat, cold, and different developmental stages or tissues. Overall, our study provides structural and molecular insights into LlWRKY genes for use in the genetic engineering in Lilium against Botrytis disease.

Two actions of juvenile hormone on the follicle cells of Rhodnius prolixus Stål

1975 ◽  
Vol 53 (8) ◽  
pp. 1187-1188 ◽  
Author(s):  
Randa Abu-Hakima ◽  
K. G. Davey
Keyword(s):  
Juvenile Hormone ◽  
Developmental Stages ◽  
Normal Animal ◽  
Rhodnius Prolixus ◽  
Follicle Cells ◽  
Follicular Epithelium ◽  
Intercellular Spaces

The follicular epithelium of vitellogenic oocytes from allatectomized females of Rhodnius fails to develop large intercellular spaces when exposed to juvenile hormone (JH) in vitro. This suggests that in the normal animal, the follicle cells require JH at two developmental stages. Differentiation of the cells in the presence of JH represents one requirement, and only those cells which have undergone this initial priming are fully competent to exhibit the second response, the development of intercellular spaces.

scholarly journals Translation initiation in Drosophila melanogaster is reduced by mutations upstream of the AUG initiator codon.

1991 ◽  
Vol 11 (4) ◽  
pp. 2149-2153 ◽  
Author(s):  
Y Feng ◽  
L E Gunter ◽  
E L Organ ◽  
D R Cavener
Keyword(s):  
Translation Initiation ◽  
Larval Stage ◽  
Developmental Stages ◽  
Germ Line ◽  
Adult Stage ◽  
Mutant Gene ◽  
Start Codon ◽  
Fold Reduction

The importance to in vivo translation of sequences immediately upstream of the Drosophila alcohol dehydrogenase (Adh) start codon was examined at two developmental stages. Mutations were introduced into the Adh gene in vitro, and the mutant gene was inserted into the genome via germ line transformation. An A-to-T substitution at the -3 position did not affect relative translation rates of the ADH protein at the second-instar larval stage but resulted in a 2.4-fold drop in translation of ADH at the adult stage. A second mutant gene, containing five mutations in the region -1 to -9, was designed to completely block translation initiation. However, transformant lines bearing these mutations still exhibit detectable ADH, albeit at substantially reduced levels. The average fold reduction at the second-instar larval stage was 5.9, while at the adult stage a 12.5-fold reduction was observed.

Applications of Advanced Nanotechnology in Stem Cell Research

2021 ◽  
Vol 13 (2) ◽  
pp. 188-198
Author(s):  
Chih-Hui Yang ◽  
Shu-Ling Huang ◽  
Yi-Ting Wang ◽  
Chun-Ho Chang ◽  
Ya-Chi Tsai ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Research ◽  
Biomedical Applications ◽  
Academic Research ◽  
Review Article ◽  
Technical Problems ◽  
Biomedical Industry ◽  
Significant Attention

Nanotechnology gives rise to new breakthroughs and developments in various fields. The applications of advanced nanotechnology may resolve the current technical problems encountered in stem cell research. Nanotechnology has gained significant attention in both academic research and the biomedical industry in recent years. In this mini-review article, the progress of nanotechnology-aided stem cell studies has been surveyed, and the in vitro and in vivo applications of nanotechnology have been introduced. The in vitro studies are divided into three categories: isolation, detection, and regulation. The progress of in vivo studies and trends in biomedical applications have also been addressed.

Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes

Development ◽  
1998 ◽  
Vol 125 (12) ◽  
pp. 2273-2282 ◽  
Author(s):  
W. Dean ◽  
L. Bowden ◽  
A. Aitchison ◽  
J. Klose ◽  
T. Moore ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Es Cells ◽  
Embryonic Stem ◽  
Upstream Region ◽  
Imprinted Genes ◽  
Epigenetic Alterations ◽  
Es Cell ◽  
Biallelic Expression

In vitro manipulation of preimplantation mammalian embryos can influence differentiation and growth at later stages of development. In the mouse, culture of embryonic stem (ES) cells affects their totipotency and may give rise to fetal abnormalities. To investigate whether this is associated with epigenetic alterations in imprinted genes, we analysed two maternally expressed genes (Igf2r, H19) and two paternally expressed genes (Igf2, U2af1-rs1) in ES cells and in completely ES cell-derived fetuses. Altered allelic methylation patterns were detected in all four genes, and these were consistently associated with allelic changes in gene expression. All the methylation changes that had arisen in the ES cells persisted on in vivo differentiation to fetal stages. Alterations included loss of methylation with biallelic expression of U2af1-rs1, maternal methylation and predominantly maternal expression of Igf2, and biallelic methylation and expression of Igf2r. In many of the ES fetuses, the levels of H19 expression were strongly reduced, and this biallelic repression was associated with biallellic methylation of the H19 upstream region. Surprisingly, biallelic H19 repression was not associated with equal levels of Igf2 expression from both parental chromosomes, but rather with a strong activation of the maternal Igf2 allele. ES fetuses derived from two of the four ES lines appeared developmentally compromised, with polyhydramnios, poor mandible development and interstitial bleeding and, in chimeric fetuses, the degree of chimerism correlated with increased fetal mass. Our study establishes a model for how early embryonic epigenetic alterations in imprinted genes persist to later developmental stages, and are associated with aberrant phenotypes.

An in vitro bioassay for a mulluscan gonadotropin

1975 ◽  
Vol 62 (2) ◽  
pp. 433-446
Author(s):  
M. J. Wells ◽  
R. K. O'Dor ◽  
S. K. Buckley
Keyword(s):  
Protein Synthesis ◽  
High Rate ◽  
Follicle Cells ◽  
Octopus Vulgaris ◽  
Amino Acid Incorporation ◽  
Gland Extract ◽  
Acid Incorporation ◽  
Kinetics Of

1. Protein synthesis occurs at a high rate in the ovaries of maturing Octopus vulgaris and can be measured from the incorporation of [14C]leucine in vivo and in isolated groups of eggs in vitro. 2. Removal of the optic glands in vivo 1--3 days prior to testing markedly reduces amino acid incorporation in vivo or in vitro. After 5 days in vivo incorporation stops. 3. The rate of incorporation in vitro is increased by the addition of optic gland extract. 4. Analysis of the kinetics of leucine uptake and incorporation in vitro indicates that the hormone has an effect on the inward transport of leucine which is independent of its action on protein synthesis. 5. Electron-microscope studies of the follicle cells and ova show that the former are the site of protein synthesis. 6. Changes in either uptake or incorporation into protein by the follicle cells can be used as a qualitative biolobical assay for the optic gland hormone. Uptake is very easy to measure but incorporation is the more sensitive parameter. Either is potentially suitable as a quantitative assay for this and perhaps also for other molluscan gonadotropins.

Calreticulin Mediates Anesthetic Sensitivity in Drosophila melanogaster 

2003 ◽  
Vol 99 (4) ◽  
pp. 867-875 ◽  
Author(s):  
Sumiko Gamo ◽  
Junya Tomida ◽  
Katsuyuki Dodo ◽  
Dai Keyakidani ◽  
Hitoshi Matakatsu ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Double Mutant ◽  
Developmental Stages ◽  
Northern Blotting ◽  
General Anesthetics ◽  
Wild Type ◽  
Mutant Strains ◽  
Low Expression

Background Various species, e.g., Caenorhabditis elegans, Drosophila melanogaster, and mice, have been used to explore the mechanisms of action of general anesthetics in vivo. The authors isolated a Drosophila mutant, ethas311, that was hypersensitive to diethylether and characterized the calreticulin (crc) gene as a candidate of altered anesthetic sensitivity. Methods Molecular analysis of crc included cloning and sequencing of the cDNA, Northern blotting, and in situ hybridization to accomplish the function of the gene and its mutation. For anesthetic phenotype assay, the 50% anesthetizing concentrations were determined for ethas311, revertants, and double-mutant strains (wild-type crc transgene plus ethas311). Results Expression of the crc 1.4-kb transcript was lower in the mutant ethas311 than in the wild type at all developmental stages. The highest expression at 19 h after pupation was observed in the brain of the wild type but was still low in the mutant at that stage. The mutant showed resistance to isoflurane as well as hypersensitivity to diethylether, whereas it showed the wild phenotype to halothane. Both mutant phenotypes were restored to the wild type in the revertants and double-mutant strains. Conclusion ethas311 is a mutation of low expression of the Drosophila calreticulin gene. The authors demonstrated that hypersensitivity to diethylether and resistance to isoflurane are associated with low expression of the gene. In Drosophila, calreticulin seems to mediate these anesthetic sensitivities, and it is a possible target for diethylether and isoflurane, although the predicted anesthetic targets based on many studies in vitro and in vivo are the membrane proteins, such as ion channels and receptors.

scholarly journals The concentration of B52, an essential splicing factor and regulator of splice site choice in vitro, is critical for Drosophila development.

1994 ◽  
Vol 14 (8) ◽  
pp. 5360-5370 ◽  
Author(s):  
M E Kraus ◽  
J T Lis
Keyword(s):  
Alternative Splice ◽  
Splice Site ◽  
Developmental Stages ◽  
Immunoblot Analysis ◽  
Splicing Factor ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Alternative Splice Site

B52 is a Drosophila melanogaster protein that plays a role in general and alternative splicing in vitro. It is homologous to the human splicing factor ASF/SF2 which is essential for an early step(s) in spliceosome assembly in vitro and also regulates 5' and 3' alternative splice site choice in a concentration-dependent manner. In vitro, B52 can function as both a general splicing factor and a regulator of 5' alternative splice site choice. Its activity in vivo, however, is largely uncharacterized. In this study, we have further characterized B52 in vivo. Using Western blot (immunoblot) analysis and whole-mount immunofluorescence, we demonstrate that B52 is widely expressed throughout development, although some developmental stages and tissues appear to have higher B52 levels than others do. In particular, B52 accumulates in ovaries, where it is packaged into the developing egg and is localized to nuclei by the late blastoderm stage of embryonic development. We also overexpressed this protein in transgenic flies in a variety of developmental and tissue-specific patterns to examine the effects of altering the concentration of this splicing factor in vivo. We show that, in many cell types, changing the concentration of B52 adversely affects the development of the organism. We discuss the significance of these observations with regard to previous in vitro results.

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