The effect of osmolarity on mouse parthenogenesis

Development ◽  
1974 ◽  
Vol 31 (2) ◽  
pp. 513-526
Author(s):  
M. H. Kaufman ◽  
M. A. H. Surani
Keyword(s):  
Protein Synthesis ◽  
Culture Medium ◽  
Culture Media ◽  
Polar Body ◽  
Amino Acid Mixture ◽  
Culture Conditions ◽  
Follicle Cells ◽  
Acid Mixture ◽  
Second Polar Body

Eggs from (C57B1 × A2G)F1 mice were activated by treatment with hyaluronidase, which removed the follicle cells, and cultured in vitro. Observations were made 6–8 h after hyaluronidase treatment to determine the frequency of activation and the types of parthenogenones induced. Cumulus-free eggs resulting from hyaluronidase treatment were incubated for 2¼ h in culture media of various osmolarities. The frequency of activation was found to be dependent on the postovulatory age of oocytes, while the types of parthenogenones induced were dependent on the osmolarity of the in vitro culture medium and their postovulatory age. Culture in low osmolar medium suppressed the extrusion of the second polar body (2PB). This decreased the incidence of haploid eggs with a single pronucleus and 2PB and immediately cleaved eggs from 97·5% to 42·3% of the activated population. Where 2PB extrusion had been suppressed, 97·4% of parthenogenones contained two haploid pronuclei. Very few were observed with a single and presumably diploid pronucleus. Serial observations from 11 to 18 h after hyaluronidase treatment were made on populations of activated eggs as they entered the first cleavage mitosis after 2¼ h incubation in medium either of normal (0·287 osmol) or low (0·168 osmol) osmolarity. A delay in the time of entry into the first cleavage mitosis similar to the duration of incubation in low osmolar medium was observed. Further, eggs were incubated in control and low osmolar culture media containing uniformly labelled [U-14C]amino acid mixture to examine the extent of protein synthesis in recently activated eggs subjected to these culture conditions. An hypothesis is presented to explain the effect of incubation in low osmolar culture medium in delaying the first cleavage mitosis.

Effect of Dietary Protein and Amino Acid Mixture on Protein Synthesis in vitro in Rat Liver

1974 ◽  
Vol 16 (6) ◽  
pp. 325-336 ◽  
Author(s):  
Alexandra von der Decken ◽  
Per T. Omstedt
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Rat Liver ◽  
Dietary Protein ◽  
Amino Acid Mixture ◽  
Acid Mixture

L-methionine uptake, incorporation and effects on proliferative activity and protein synthesis in bovine claw tissue explants in vitro

2007 ◽  
Vol 146 (1) ◽  
pp. 103-115 ◽  
Author(s):  
N. L. HEPBURN ◽  
C. H. KNIGHT ◽  
C. J. WILDE ◽  
K. A. K. HENDRY ◽  
H. GALBRAITH
Keyword(s):  
Protein Synthesis ◽  
Culture Medium ◽  
Culture Media ◽  
Tissue Level ◽  
Normal Function ◽  
Regulation Of Proliferation ◽  
Methionine Uptake ◽  
Dna And Protein Synthesis

SUMMARYL-methionine is a sulphur-containing nutritionally essential amino acid. It has a number of important roles in epidermal and dermal tissues of the integument of animals. Failure of normal function of these tissues in the hoof (claw) is a cause of lameness in cattle. Little is known about quantitative relationships between post-absorptive concentrations of nutrients including sulphur-containing amino acids and uptake and utilization by epidermis and dermis of the bovine claw. These parameters were studied at the tissue level by use of an established in vitro claw explant system using tissue from cattle of beef or dairy origin and L-[35S]-labelled methionine as tracer. The results showed that uptake of L-methionine by freshly prepared solear explants in Dulbecco's Modified Eagle Medium/F-12 Nutrient Mix (DMEM/F12) (1:1) medium containing 1·0 mmol L-methionine/litre was concentrative after 5–8 min, essentially linear for up to 10 min and became curvilinear thereafter. Maximum uptake and steady state conditions were obtained at approximately 30 min. Further measurements were made following 21 h incubation in culture medium. Under conditions of varying concentrations of L-methionine and measurement of uptake after 30 min, the presence of a saturable curve, that obeyed Michaelis–Menten kinetics, was demonstrated. Values of 3·61 mmol/litre and 5·84 mmol/kg intracellular water/30 min were obtained for KM and Vmax, respectively. Uptake was not influenced by L-cysteine and L-cystine concentrations in the culture media.Similar culture and incubation conditions were used in subsequent studies of DNA and protein synthesis. These showed that rates of incorporation of L-methionine into protein fractions and stimulation of DNA synthesis measured by methyl-thymidine incorporation were dependent on L-methionine concentrations in the medium. Maximal rates occurred at approximately 50 μmol/litre, which is in the normal physiological range, and at 1% of maximum uptake capacity. Examination of histological sections by autoradiography showed localization of L-[35S]-labelled methionine in basal and suprabasal epidermal cells with limited retention in dermis. Measurement, by a range of histological, immunohistochemical, electrophoretic, western blotting and autoradiographic techniques, provided further evidence of L-methionine-dependent regulation of proliferation, differentiation and synthesis of proteins under physiological concentrations, by epidermal horn-forming cells.A key role for L-methionine is suggested in the production of horn in bovine claw. The extrapolation of these in vitro data provides guidance for strategies to optimize methionine supply to claw tissues in vivo. Such extrapolation suggests the appropriateness of delivery of systemic concentrations of 50 μmol L-methionine/litre to maximize proliferative and protein depositional activity in solear epidermis and dermis in vivo.

Timing of the First Cleavage Division of Haploid Mouse Eggs, and the Duration of its Component Stages

1973 ◽  
Vol 13 (2) ◽  
pp. 553-566 ◽  
Author(s):  
M. H. KAUFMAN
Keyword(s):  
Polar Body ◽  
Time Lapse ◽  
Follicle Cells ◽  
Cleavage Division ◽  
Cell Stage ◽  
Cell Embryo ◽  
Second Polar Body ◽  
Polar Body Extrusion ◽  
Mouse Eggs

Mouse eggs were activated by treatment with hyaluronidase which removed the follicle cells, followed by culture in vitro, and examined at the first cleavage mitosis. Second polar body extrusion usually occurred and haploid parthenogenesis was initiated. Air-dried chromosome preparations were made between 11 and 15.5 h after activation. Out of the 308 eggs examined 74 had already progressed to the 2-cell stage; the remaining 234 at the 1-cell stage were examined in detail. All chromosome preparations of the first cleavage mitosis were classified into groups corresponding with the stages of prometaphase, metaphase (early or ‘pre-chromatid’, ‘chromatid’ and ‘late chromatid’) and anaphase. An indirect estimate was made of the duration of the first cleavage mitosis and of its component stages from the incidence of stages observed at different time intervals after activation. Similar eggs were also observed at 37 °C by time-lapse cine-photography and the interval between the disappearance of the pronucleus to the beginning of telophase of the first cleavage division was determined. The results of timing studies on the haploid eggs were compared with results obtained from similar observations on the first cleavage division of fertilized eggs which would of course normally be diploid. Artificially activated eggs with 2 pronuclei, resulting from second polar body suppression, were also examined, and serial chromosome preparations during mitosis showed that the 2 pronuclear chromosome groups unite on the first cleavage spindle and divide to give a hetero-zygous diploid 2-cell embryo.

291 EFFECTS OF ANTIOXIDANT AND CULTURE MEDIUM ON THE DEVELOPMENT OF PORCINE IN VITRO-MATURED - IN VITRO-FERTILIZED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 261
Author(s):  
C. Choe ◽  
D.-S. Son ◽  
S.-H. Choi ◽  
S.-R. Cho ◽  
H.-J. Kim ◽  
...  
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Culture Conditions ◽  
Free Oxygen Radicals ◽  
Blastocyst Formation ◽  
Free Oxygen ◽  
Statistical Analysis System ◽  
Developmental Rates ◽  
Analysis System

Most cells cultured in vitro are exposed to the risk of injury by free oxygen radicals (FOR). However, some of FOR-induced injury could be reduced by the antioxidants and culture medium used for in vitro embryos. This study was undertaken to examine the effects of the antioxidant and culture medium on the development of porcine in vitro-matured–in vitro-fertilized embryos. In Experiment 1, we treated the porcine oocytes in NCSU23 medium with various concentrations of β-mercaptoethanol (β-ME) to determine the effective concentration of antioxidants during IVM of porcine oocytes. In Experiment 2, we tested different culture media to find the proper culture conditions for in vitro porcine embryos. The porcine oocytes that were matured in NCSU23 medium and then fertilized in mTBM medium were cultured in NCSU23 or porcine zygote medium-5. All steps (maturation, fertilization, and development) were carried out in vitro. Differences were analyzed among treatments using the general linear model (GLM) procedure in the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). The results were summarized as follows. Various concentrations of β-ME showed different developmental rates in porcine embryos. The rates of blastocyst formation at Day 7 after IVF were 9.2 � 1.8 (n = 65), 10.0 � 4.2 (n = 80), 17.5 � 1.1 (n = 63), 20.7 � 1.7 (n = 82), and 14.6 � 1.4 (n = 82) in oocytes treated with β-ME at 0, 10, 25, 50, and 100 �M during IVM, respectively. Of the concentrations of β-ME tested, 50 �M β-ME markedly increased the rates of blastocyst formation at Day 7 (P < 0.05). The rates of blastocyst formation at Day 7 in the NCSU23 and PZM-5 culture media of porcine IVF-derived embryos were 18.8 � 2.6 (n = 96) and 15.6 � 7.1 (n = 77), respectively. The developmental rates were slightly increased in NCSU23, compared with those in PZM-5, but there were no significant differences (P < 0.05) between the NCSU23 and PZM-5 media. In conclusion, these results suggest that the addition of 50 �M β-ME in the IVM medium can improve developmental the rates of porcine embryos in vitro.

In vitro activation of mouse eggs

Development ◽  
1974 ◽  
Vol 31 (2) ◽  
pp. 497-512
Author(s):  
C. F. Graham ◽  
Z. A. Deussen
Keyword(s):  
Dna Synthesis ◽  
Culture Medium ◽  
Tritiated Thymidine ◽  
Polar Body ◽  
Similar Time ◽  
Body Formation ◽  
Polar Body Formation ◽  
Second Polar Body ◽  
Mouse Eggs

Unfertilized mouse eggs were activated in vitro with hyaluronidase. Subsequently they were exposed to culture medium at different osmolarities. In full strength White's culture medium they tended to form one pronucleus and a second polar body. The majority of these eggs were haploid. In 4/5 and 3/5 dilutions of this medium, the second polar body formation was suppressed and eggs tended to form with one or two pronuclei. Those with one pronucleus were diploid and those with two pronuclei could either form a diploid or form a haploid mosaic. Old eggs tended to immediately cleave and form haploid mosaics. DNA synthesis was studied in activated eggs using tritiated thymidine and autoradiography. DNA synthesis occurred at a similar time in fertilized and activated eggs.

The effect of strain of donor eggs, culture conditions, and experimental protocol on release of eggshell calcium by the chick chorioallantoic membrane in vitro

1997 ◽  
Vol 75 (9) ◽  
pp. 1474-1478 ◽  
Author(s):  
Mary J. Packard
Keyword(s):  
Culture Medium ◽  
Calcium Release ◽  
Culture Media ◽  
Chorioallantoic Membrane ◽  
Culture Conditions ◽  
Factors Affecting ◽  
Chick Chorioallantoic Membrane ◽  
Contrast Exposure ◽  
Donor Eggs

Factors affecting release of calcium by the chick chorioallantoic membrane in vitro were assessed by culturing explants of shell with and without the chorioallantoic epithelium. Explants were removed from eggs on day 16 of incubation and cultured using techniques similar to those used to examine resorption of bone in vitro. The region of an egg from which an explant is removed, the strain of donor eggs used, and addition of bovine serum albumin to the culture medium did not affect release of calcium. In contrast, exposure of explants to a variety of different culture media (DMEM, Medium 199, BGJb, and RPMI) introduced considerable variation in calcium release. Calcium release in vitro was also examined by removing the blunt end of a fertile egg as well as the embryo and yolk. The remaining preparation was filled with culture medium, covered, and cultured for two 24-h periods, with a change of medium after the first 24 h. Preparations from which the chorioallantoic membrane had been removed released very little calcium during culture, whereas those with the epithelium in place released considerable quantities. However, release of calcium by preparations with the membrane declined during the second interval. When surface area is taken into account, such preparations mobilize less calcium than explants cultured under similar conditions.

scholarly journals Improving In Vitro Culture of Human Male Fetal Germ Cells

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2033
Author(s):  
Myriam Martin-Inaraja ◽  
Monica Ferreira ◽  
Jasin Taelman ◽  
Cristina Eguizabal ◽  
Susana M. Chuva De Sousa Lopes
Keyword(s):  
Germ Cells ◽  
Culture Medium ◽  
Culture Media ◽  
Culture Conditions ◽  
Pronounced Difference ◽  
Coated Substrate ◽  
Male Gametes ◽  
The Media

Male human fetal germ cells (hFGCs) give rise to spermatogonial stem cells (SSCs), which are the adult precursors of the male gametes. Human SSCs are a promising (autologous) source of cells for male fertility preservation; however, in contrast to mouse SSCs, we are still unable to culture them in the long term. Here, we investigated the effect of two different culture media and four substrates (laminin, gelatin, vitronectin and matrigel) in the culture of dissociated second trimester testes, enriched for hFGCs. After 6 days in culture, we quantified the presence of POU5F1 and DDX4 expressing hFGCs. We observed a pronounced difference in hFGC number in different substrates. The combination of gelatin-coated substrate and medium containing GDNF, LIF, FGF2 and EGF resulted in the highest percentage of hFGCs (10% of the total gonadal cells) after 6 days of culture. However, the vitronectin-coated substrate resulted in a comparable percentage of hFGCs regardless of the media used (3.3% of total cells in Zhou-medium and 4.8% of total cells in Shinohara-medium). We provide evidence that not only the choices of culture medium but also choices of the adequate substrate are crucial for optimizing culture protocols for male hFGCs. Optimizing culture conditions in order to improve the expansion of hFGCs will benefit the development of gametogenesis assays in vitro.

scholarly journals PROTEIN SYNTHESIS IN SYNAPTOSOMAL FRACTIONS

1972 ◽  
Vol 52 (3) ◽  
pp. 526-535 ◽  
Author(s):  
P. Gambetti ◽  
L. A. Autilio-Gambetti ◽  
N. K. Gonatas ◽  
B. Shafer
Keyword(s):  
Protein Synthesis ◽  
Specific Activity ◽  
Mitochondrial Protein ◽  
Amino Acid Mixture ◽  
Acid Mixture ◽  
Mitochondrial Protein Synthesis ◽  
Synaptosomal Fraction ◽  
Tritiated Amino Acids ◽  
Vitro Protein

A quantitative ultrastructural radioautographic study of in vitro protein synthesis has been carried out in rat synaptosomal fractions incubated with tritiated leucine or a tritiated amino acid mixture. Analysis of grain density distribution demonstrated that presynaptic endings are labeled. 30–50% of the developed grains, representing tritiated amino acids incorporated into proteins, were related to presynaptic endings which accounted for 75–77% of the total processes. 34–45% of the grains were related to processes containing ribosomes which accounted for only 4–7% of the total processes. The relative specific activity of these ribosome-containing processes, some of which could be identified as postsynaptic elements, was up to ten times higher than that of the presynaptic ending. These findings indicate that protein synthesis takes place in vitro in presynaptic terminals although to a significantly lesser degree than that occurring in ribosome-containing processes, which, with other nonpresynaptic processes, are at the present time unavoidable contaminants of synaptosomal fractions. Presynaptic endings that in radioautographs contained no mitochondria were labeled. Also, presynaptic endings were labeled after incubation in the presence of chloramphenical which inhibited 20% of the protein synthesis of the synaptosomal fraction. It is concluded that besides mitochondrial protein synthesis, another protein synthesizing system operates in presynaptic endings in vitro.

scholarly journals PROTEIN SYNTHESIS BY ISOLATED PEA NUCLEOLI

1963 ◽  
Vol 18 (1) ◽  
pp. 41-50 ◽  
Author(s):  
Max L. Birnstiel ◽  
Beal B. Hyde
Keyword(s):  
Protein Synthesis ◽  
Amino Acid Mixture ◽  
Microscopic Investigation ◽  
Electron Microscopic Investigation ◽  
Nucleolar Protein ◽  
Acid Mixture ◽  
Electron Microscopic ◽  
Submicroscopic Structure ◽  
Acid And Base

A new method is described for the preparation of active, nucleus-free nucleoli and chromatin in relatively high purity and in sufficient quantities to permit biochemical and electron microscopic investigation. This method consists of disintegrating previously isolated nuclei by grinding with glass beads in an isotonic medium thus liberating structurally intact nucleoli and chromatin threads. Nucleoli and chromatin are then purified by differential centrifugation in Ficoll solutions. A study of the chemical composition, submicroscopic structure, and biological activity of the nucleolar preparation has been made. An equivalent study of the chromatin material has also been carried out in order to assess the significance of chromosomal contamination in nucleolar protein synthesis. The isolated nucleoli rapidly incorporate leucine-C14 into acid and base stable compounds in vitro. Such incorporation lasts for 20 minutes at 37°C and is enhanced by the addition of an energy-regenerating system and a complete amino acid mixture. It is independent of the nuclear Ph 5 enzymes. The bulk of the incorporated label is recovered in the residual, ribosome-like nucleolar protein fraction and a small percentage is found in the acid-extractable basic proteins. The rate of protein synthesis by isolated nucleoli is more rapid than that occurring in the chromatin fraction. This is taken as an additional proof that the nucleolus is the principal site of protein synthesis in the interphase pea nucleus.

Some features of clonal micropropagation of decorative cultivars of Amelanchier Medik.

2020 ◽  
pp. 97-104
Author(s):  
E. N. Raeva-Bogoslovskaya ◽  
O. I. Molkanova
Keyword(s):  
In Vitro Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Culture Conditions ◽  
Clonal Micropropagation ◽  
Morphogenetic Potential

In vitro culture conditions were optimized for representatives of the genus Amelanchier Medik. at the stages of micropropagation and rooting. A significant effect of the mineral and hormonal compositions of culture media on the morphogenetic potential of the cultivars of serviceberry has been established. The use at the stage of micropropagation of the MS culture medium with addition 1,0 mg / L 6-benzylaminopurine (6-BAP) promoted the active microshoot regeneration of the studied genotypes. For the induction of rhizogenesis the type of auxin as IB A at a concentration of 1,0 mg / L was used.

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