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.

Induction of triploidy in the mouse by cytochalasin B

Development ◽  
1975 ◽  
Vol 34 (2) ◽  
pp. 279-289
Author(s):  
Anna Niemierko
Keyword(s):  
Culture Medium ◽  
Cytochalasin B ◽  
Sex Chromosome ◽  
Polar Body ◽  
Cell Number ◽  
Second Polar Body ◽  
And Control ◽  
Mouse Eggs

Mouse eggs fertilized in vivo were treated with cytochalasin B in vitro (5 μg/ml of culture medium) at he moment of extrusion of the second polar body (2·5, 3·0, 3·5 h after copulation). Cytochalasin B inhibits cytokinesis of the second maturation division, so that triploid digynic eggs are formed in over 50% of treated eggs. Triploid eggs were transplanted to the oviducts of recipients. On the 4th and 5th day of development 41·7% of transplanted eggs were recovered. All embryos recovered on the 4th day were morulae, while on the 5th day blastocysts predominated. Recovered embryos were studied for cell number and ploidy. Twenty-three of 27 embryos with analysable metaphase plates were triploid and four were diploid (the latter were found in females into which both triploid and control diploid eggs were transplanted). Sex chromosome constitution was determined in seven cases: four triploids were XXY and three were XXX.

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.

Preimplantation development of gynogenetic diploid mouse embryos

Development ◽  
1982 ◽  
Vol 69 (1) ◽  
pp. 215-222
Author(s):  
Ewa Borsuk
Keyword(s):  
Cytochalasin B ◽  
Polar Body ◽  
Mouse Embryos ◽  
Fertilization In Vitro ◽  
Body Formation ◽  
Male Pronucleus ◽  
Step Procedure ◽  
Gynogenetic Diploid ◽  
Polar Body Formation

Diploid gynogenetic mouse embryos were produced in a three-step procedure: fertilization in vitro, suppression of the 2nd polar body formation by Cytochalasin B, and microsurgical removal of the male pronucleus. The operated eggs were transplanted to the oviduct of recipient females for 72 or 96 h. The overall recovery rate was 73%, but compacted morulae and blastocysts constituted only 28·6% of transplanted eggs. After 72 h blastocysts were rare (3·5%) but 24 h later their incidence increased to 21·2%. In eggs homozygous for T6 chromosome it was possible to prove karyologically that the male pronucleus was effectively removed and that the diploid genome was of purely maternal origin.

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.

Memoirs: The Germ-Cell Cycle in Phytophaga destructor Say

1935 ◽  
Vol s2-77 (308) ◽  
pp. 585-604
Author(s):  
MARGOT E. METEALFE
Keyword(s):  
Germ Cells ◽  
Growth Stage ◽  
Germ Line ◽  
Polar Body ◽  
Metaphase Plate ◽  
Body Formation ◽  
Polar Bodies ◽  
Complicated Process ◽  
Female Pronucleus ◽  
Polar Body Formation

1. The somatic cells in both sexes of Phytophaga destructor Say contain four pairs of V-shaped chromosomes, the sex-group being indistinguishable in size or form. 2. The germ-cells in both sexes contain eight pairs of chromosomes. 3. The maturation of the egg follows the normal course of development, eight bivalents being formed. After polar body formation the female pronucleus has eight chromosomes. The polar bodies are never extruded from the egg. 4. Spermatogenesis is a complicated process, the details of which have not been satisfactorily determined. The growth stage appears to take place before the last spermatogonial division. No pairing of chromosome has been observed, and apparently no metaphase plate is formed at meiosis. Eeduction is effected by the expulsion of two buds each containing four chromosomes. Thus only one sperm is produced from each spermatocyte. 5. One or more sperms may enter the egg at fertilization. 6. The germ-line is differentiated from the soma at the eightcell stage. 7. At the fifth cleavage the somatic nuclei eliminate half their number of chromosomes, and are left with eight chromosomes. 8. Migration of the germ nuclei takes place at the sixteencell stage. 9. The relation of the chromosome numbers in the somatic and germ lines is discussed.

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