Growth in vitro and acquisition of meiotic competence after the cryopreservation of isolated mouse primary ovarian follicles

1991 ◽  
Vol 3 (5) ◽  
pp. 593 ◽  
Author(s):  
J Carroll ◽  
DG Whittingham ◽  
MJ Wood
Keyword(s):  
Subsequent Development ◽  
Collagen Gels ◽  
Freezing And Thawing ◽  
Oocyte Growth ◽  
Large Numbers ◽  
Mean Diameter ◽  
Old Mice ◽  
Meiotic Competence

The growth and acquisition of meiotic competence of oocytes from fresh and frozen-thawed primary follicles collected from 10-day-old mice was compared during culture in collagen gels for 12 days. The oocytes contained in primary follicles have a mean diameter of about 48 microns and do not resume meiosis without further growth and development. During the 12-day culture period the mean diameter of the oocytes increased to over 60 microns. The oocytes were capable of resuming meiosis when isolated from the gel and cultured in the absence of follicular cells in a manner similar to that observed in vivo. Freezing and thawing did not affect oocyte growth or the ability to resume meiosis; this demonstrates the possibility of storing large numbers of female gametes for subsequent development.

scholarly journals Zinc supports transcription and improves meiotic competence of growing bovine oocytes

Reproduction ◽  
2020 ◽  
Vol 159 (6) ◽  
pp. 679-691
Author(s):  
Valentina Lodde ◽  
Rodrigo Garcia Barros ◽  
Priscila Chediek Dall’Acqua ◽  
Cecilia Dieci ◽  
Claude Robert ◽  
...  
Keyword(s):  
Culture Medium ◽  
Zinc Supplementation ◽  
Oocyte Growth ◽  
Zinc Chelator ◽  
Bovine Oocytes ◽  
Meiotic Competence ◽  
Medium Supplementation

In the last years, many studies focused on the understanding of the possible role of zinc in the control of mammalian oogenesis, mainly on oocyte maturation and fertilization. However, little is known about the role of zinc at earlier stages, when the growing oocyte is actively transcribing molecules that will regulate and sustain subsequent stages of oocyte and embryonic development. In this study, we used the bovine model to gain insights into the possible involvement of zinc in oocyte development. We first mined the EmbryoGENE transcriptomic dataset, which revealed that several zinc transporters and methallothionein are impacted by physiological conditions throughout the final phase of oocyte growth and differentiation. We then observed that zinc supplementation during in vitro culture of growing oocytes is beneficial to the acquisition of meiotic competence when subsequently subjected to standard in vitro maturation. Furthermore, we tested the hypothesis that zinc supplementation might support transcription in growing oocytes. This hypothesis was indirectly confirmed by the experimental evidence that the content of labile zinc in the oocyte decreases when a major drop in transcription occurs in vivo. Accordingly, we observed that zinc sequestration with a zinc chelator rapidly reduced global transcription in growing oocytes, which was reversed by zinc supplementation in the culture medium. Finally, zinc supplementation impacted the chromatin state by reducing the level of global DNA methylation, which is consistent with the increased transcription. In conclusion, our study suggests that altering zinc availability by culture-medium supplementation supports global transcription, ultimately enhancing meiotic competence.

Animal Models and Alternatives in the Quality Control of Vaccines: Are In Vitro Methods or In Vivo Methods the Scientific Equivalent of the Emperor's New Clothes?

1995 ◽  
Vol 23 (1) ◽  
pp. 61-73
Author(s):  
Coenraad Hendriksen ◽  
Johan van der Gun
Keyword(s):  
Quality Control ◽  
Test Methods ◽  
In Vitro Test ◽  
Large Numbers ◽  
Alternative Approach ◽  
Inactivated Vaccines ◽  
Vitro Test

In the quality control of vaccine batches, the potency testing of inactivated vaccines is one of the areas requiring very large numbers of animals, which usually suffer significant distress as a result of the experimental procedures employed. This article deals with the potency testing of diphtheria and tetanus toxoids, two vaccines which are used extensively throughout the world. The relevance of the potency test prescribed by the European Pharmacopoeia monographs is questioned. The validity of the potency test as a model for the human response, the ability of the test to be standardised, and the relevance of the test in relation to the quality of the product are discussed. It is concluded that the potency test has only limited predictive value for the antitoxin responses to be expected in recipients of these toxoids. An alternative approach for estimating the potency of toxoid batches is discussed, in which a distinction is made between estimation of the immunogenic potency of the first few batches obtained from a seed lot and monitoring the consistency of the quality of subsequent batches. The use of animals is limited to the first few batches. Monitoring the consistency of the quality of subsequent batches is based on in vitro test methods. Factors which hamper the introduction and acceptance of the alternative approach are considered. Finally, proposals are made for replacement, reduction and/or refinement (the Three Rs) in the use of animals in the routine potency testing of toxoids.

Activation of the peptidergic neurosecretory system in Diphyllobothrium dendriticum (Cestoda: Pseudophyllidea)

Parasitology ◽  
1981 ◽  
Vol 83 (2) ◽  
pp. 243-247 ◽  
Author(s):  
Margaretha K. S. Gustafsson ◽  
Marianne C. Wikgren
Keyword(s):  
Neurosecretory Cells ◽  
Final Host ◽  
Fish Host ◽  
Neurosecretory System ◽  
Weak Reaction ◽  
Diphyllobothrium Dendriticum ◽  
Large Numbers ◽  
Short Times

SUMMARYThe activation of the peptidergic neurosecretory system in Diphyllobothrium dendriticum was studied following cultivation of plerocercoids for short times in vitro and in vivo. In the plerocercoid the neurosecretory cells gave a very weak reaction with paraldehyde fuchsin (PAF). After cultivation for 1 h large numbers of neurosecretory cells filled with PAF-positive granules were evident. The significance of the activation of the neurosecretory system during the transfer of the worm from the cold-blooded fish host to the warm-blooded final host is discussed.

Studies on the functional activity of organotypically cultured mouse ovary

Development ◽  
1966 ◽  
Vol 15 (2) ◽  
pp. 133-141
Author(s):  
T. N. Chapekar ◽  
G. V. Nayak ◽  
Kamal J. Ranadive
Keyword(s):  
Functional Activity ◽  
Synthetic Medium ◽  
Culture Conditions ◽  
Short Term ◽  
Plasma Clot ◽  
Rat Ovary ◽  
Old Mice ◽  
Term Maintenance

Short-term maintenance of mouse and rat ovary in organotypic culture system is no longer a problem (Martinovitch, 1938; Gaillard, 1953; Trowell, 1959). Gaillard (1953) cultivated ovaries from 7- to 8-day-old and 21-day-old mice for a week on the plasma clot. Trowell (1959) maintained ovaries of 8-day-old mice on a synthetic medium in an O2-CO2 atmosphere for 9 days. He observed no histological differentiation in the tissues of the ovary. What needs confirmation and further investigation is the possibility of maintenance of functional activity of the ovary under culture conditions. A study was therefore undertaken to investigate if an ovary, cultivated in vitro for some time, shows hormonal activity when transplanted in vivo. In the present work cultured ovaries were grafted in the anterior eye-chamber of spayed female mice and the development of secondary sex organs such as mammary glands and uterus was studied.

scholarly journals ELECTRON MICROSCOPY OF PLASMA-CELL TUMORS OF THE MOUSE

1961 ◽  
Vol 9 (2) ◽  
pp. 369-381 ◽  
Author(s):  
D. F. Parsons ◽  
M. A. Bender ◽  
E. B. Darden ◽  
Guthrie T. Pratt ◽  
D. L. Lindsley
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Complex Structure ◽  
Granular Body ◽  
Virus Particles ◽  
Culture Cells ◽  
Large Numbers ◽  
Tumor Agent

The X5563 tumor has been grown in tissue culture. Cells similar to those of the original tumor migrated from the explant and attached to the glass walls of the culture vessels. Electron microscopy showed that large numbers of particles, similar in morphology to virus particles, were associated with these cells after 7 days of culture. The two principal types of particles found in the tumor in vivo appear to be present in vitro. Many more of these particles, however, were larger and showed a more complex structure. Whereas the particles were mainly localized inside endoplasmic reticulum or the Golgi zone in the tumors in vivo, in the tissue culture the majority of the particles were associated with the plasma membrane and were found outside of the cells. The relation of the particles to the granular body is discussed as well as a possible relation to the mammary tumor agent.

scholarly journals The selective binding and transmigration of monocytes through the junctional complexes of human endothelium.

1988 ◽  
Vol 168 (5) ◽  
pp. 1865-1882 ◽  
Author(s):  
N A Pawlowski ◽  
G Kaplan ◽  
E Abraham ◽  
Z A Cohn
Keyword(s):  
Silver Staining ◽  
Umbilical Vein ◽  
Collagen Gel ◽  
Collagen Gels ◽  
Silver Stain ◽  
Term Survival ◽  
Topographical Distribution ◽  
Junctional Complexes

Human monocytes show a high affinity for vascular endothelium both in vitro and in vivo. To explore monocyte-endothelial interaction in greater detail, we have developed a new in vitro model for growth of human endothelial cells (EC). Human umbilical vein EC (HUVEC) cultured upon collagen gels form confluent monolayers of EC that bind silver at their intercellular border similar to cells in situ. Intercellular junctional structures, both adherens and tight junctions, were identified. In contrast, HUVEC grown on plastic surfaces did not stain with silver. The silver-staining characteristic of EC-collagen monolayers was reversible and related to their in vitro maturation and senescence. Silver staining of EC borders provided a grid by which the location of monocyte binding to the luminal surface of individual EC could be assessed. Using this technique, we found that monocytes preferentially bound to the margins of EC, in approximation to the silver-staining junctions. These results suggest that EC determinants recognized by monocytes occur in a unique topographical distribution on the apical face of EC. After binding, monocytes migrated through the EC monolayers at high basal rates. The lack of penetration of collagen gels in the absence of an EC monolayer suggested the generation of EC-specific chemotactic signal(s). Monocytes were observed to pass between EC without evidence of disruption of the monolayer. Silver stain remained present during all phases of migration, and under transmission electron microscopy, junctional complexes were found proximal to monocytes that had just completed their passage through the monolayer. After orientation to the basal surface of the EC monolayer, monocytes migrated randomly into the underlying collagen gel. Monocyte adherence, penetration, migration, and long term survival can be studied under these conditions.

INACTIVATION OF RENIN IN A MIXED MITOCHONDRIAL-LYSOSOMAL FRACTION OF POST-PARTUM UTERUS

1979 ◽  
Vol 92 (4) ◽  
pp. 731-745
Author(s):  
Jørgen Jørgensen
Keyword(s):  
Alkaline Phosphatase ◽  
Post Partum ◽  
Similar Rate ◽  
Freezing And Thawing ◽  
Lysosomal Fraction ◽  
Triton X

ABSTRACT The marked renin inactivation seen during in vitro incubation of post-partum uterine slices which mimics the in vivo condition, is not accompanied by a similar general proteolysis. The inactivating mechanism is so far non-specific with respect to organ or species as added hog renal renin is inactivated at a similar rate as endogenous renin. Endogenous alkaline phosphatase is not significantly inactivated and added alkaline phosphatase is completely stable. A marked inactivation of endogenous renin also takes place during incubation of a mixed mitochondrial-lysosomal suspension prepared from post-partum uterus. The process is more pronounced at pH 7.4 than at 6.8. Freezing and thawing and addition of Triton X-100 prior to incubation inhibits the inactivation. ATP and α-ketoglutarate slightly stimulates the process while CoA and chloroquine have no effect. Both iodoacetate and phenylmethylsulphonylfluoride inhibit the inactivation, suggesting that more than one enzyme is involved in the inactivation.

Association between tension and orientation of periodontal ligament fibroblasts and exogenous collagen fibres in collagen gels in vitro

1982 ◽  
Vol 58 (1) ◽  
pp. 125-138
Author(s):  
C.G. Bellows ◽  
A.H. Melcher ◽  
J.E. Aubin
Keyword(s):  
Three Dimensional ◽  
Lower Surface ◽  
Cytochalasin D ◽  
Collagen Gels ◽  
Collagen Fibres ◽  
Periodontal Ligament Fibroblasts ◽  
The Relationship ◽  
Develop Tension

The relationship between the development of tension in sheets of fibroblasts and the orientation of these cells and collagen fibres in collagen gels was examined. Cell-containing, three-dimensional collagen gels were established in agarose-coated Epon dies measuring 10 mm X 4 mm X 4 mm, to which pieces of demineralized tooth and bone had been attached at opposite ends. Contraction of the gel into an opaque structure suspended between the two particles occurred over 24 h and resulted in concave upper and lateral surfaces and a flat to slightly concave lower surface. Initial orientation of the fibres along the tooth-bone axis was followed by similar orientation of the cells. Gels cast without cells exhibited no change in dimensions. Release of the tooth particle after 12 or 24 h of incubation led to shortening of the contracted gels 5 min following release. This shortening was significantly greater (P less than 0.001) than that of uncontracted or slightly contracted gels (1 h and 3 h incubation). Gels attached at one end only compacted around the site of attachment but did not show orientation of cells or fibres. Gels containing colcemid or cytochalasin D were only slightly compacted and did not develop tension. Collagen fibres, but not cell in colcemid-containing gels, showed some alignment; neither were aligned in the presence of cytochalasin D. These data suggest that both microtubules and microfilaments are necessary for alignment of cells and the establishment of tension between two points of attachment in collagen gels. Furthermore, they lend support to our previously advanced hypothesis that the development of tension between two points can result in the orientation of the cells along an axis connecting the points of attachment. This could provide a mechanism for the development of oriented fibre systems in vivo.

Lethality of a tritiated amino acid in early mouse embryos

Development ◽  
1985 ◽  
Vol 88 (1) ◽  
pp. 209-217
Author(s):  
Janet L. Wiebold ◽  
Gary B. Anderson
Keyword(s):  
Specific Activity ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Radiation Induced ◽  
Specific Activities ◽  
Tritiated Amino Acids ◽  
Early Mouse

2- to 4-cell and morula- to blastocyst-stage mouse embryos were cultured for 1 h in tritiated leucine at two specific activities and their subsequent development followed in vitro and in vivo (after transfer to recipients), respectively. 2- to 4-cell embryos that incorporated an average of 42 d.p.m. per embryo were impaired in their ability to develop to the morula and blastocyst stage. Recipients receiving morulae and blastocysts that had incorporated an average of 384 d.p.m. per embryo failed to produce young. Reduction of the specific activity improved the viability of embryos both in vitro and in vivo but development was still less than that of unlabelled embryos. Protein degradation curves were different for both 2- to 4-cell and morulato blastocyst-stage embryos labelled at the two different specific activities. Most studies using tritiated amino acids have employed higher specific activities than those used here and they may have to be reevaluated due to the possibility of radiation-induced artifacts.

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