Diploid parthenogenetic mouse embryos produced by heat-shock and Cytochalasin B

Development ◽  
1976 ◽  
Vol 35 (1) ◽  
pp. 25-39
Author(s):  
Hanna Bałakier ◽  
Andrzej K. Tarkowski
Keyword(s):  
Heat Shock ◽  
Cytochalasin B ◽  
Polar Body ◽  
Implantation Rate ◽  
Mouse Embryos ◽  
Activation Rate ◽  
Second Polar Body ◽  
Stage Characteristic ◽  
Parthenogenetic Mouse Embryos

Swiss albino and C57BL/10 eggs from induced ovulations, and spontaneously ovulated A eggs, were activated in vitro by a heat shock of 44 °C for 5 or 7·5 min and cultured in the presence of 10 μg/ml of Cytochalasin B (CB) for 5–8 h. The activation rate was about 70 % in Swiss albino, 40 % in C57BL and 90 % in A eggs. CB suppressed second polar body (2P.B.) formation in over 90 % of activated eggs, with the majority containing two pronuclei. When eggs were placed in CB-free medium their surface became wrinkled and they formed protrusions of various sizes, which in some eggs detached to form enucleate or pronucleate cytoplasmic fragments; some eggs broke down completely into fragments. In most eggs, however, the surface smoothed out in a few hours and suppression of 2P.B. appeared to be permanent. The rate of development of these eggs after transplantation to the oviduct was delayed in terms both of cell divisions and of the time of blastocyst formation. Out of 41 implants collected on the 8th–10th day of pregnancy only two healthy looking egg-cylinders were found on the 8th and 9th day; both were retarded, at the stage characteristic for the 7th day of normal development. The reasons for delayed preimplantation development and low implantation rate are discussed. The present experiments corroborate earlier observations that parthenogenetic mouse embryos, even if diploid, rarely survive in the uterus beyond the egg-cylinder stage.

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.

Development in vitro and in vivo of aggregated parthenogenetic bovine embryos

1995 ◽  
Vol 7 (5) ◽  
pp. 1073 ◽  
Author(s):  
A Boediono ◽  
S Saha ◽  
C Sumantri ◽  
T Suzuki
Keyword(s):  
Cytochalasin B ◽  
Polar Body ◽  
Cleavage Rate ◽  
Development In Vitro ◽  
Second Polar Body ◽  
Bovine Oocytes ◽  
Longitudinal Diameter ◽  
Parthenogenetic Embryos

Mature bovine oocytes were activated with 7% ethanol followed by cytochalasin B or D treatment. Most oocytes extruded a second polar body and formed one pronucleus when treated with 7% ethanol alone [35/43 (81%)]. With ethanol followed by cytochalasin B or D, overall activation frequency was 70% (309/441), with activated oocytes containing two pronuclei. The cleavage rate was not significantly different between treatment with ethanol alone and ethanol followed by 5 micrograms mL-1 cytochalasin B, but it was significantly lower than in fertilized oocytes (P < 0.01). However, the blastocyst production rate was significantly different (P < 0.01) among the treatments. The incidence of parthenogenetic embryos with normal (diploid) complements and with chromosome anomalies (2N/4N) was 68% (17/25) and 32% (8/25) respectively, and this was not affected by cryopreservation treatment. The longitudinal diameter of aggregated-four embryos cultured in vitro was greater (P < 0.01) than aggregated-two or single embryos. One of the aggregated-four parthenogenetic embryos was further cultured in vitro and developed up to Day 27 after activation, with a diameter of 2980 microns. The aggregated-four parthenogenetic embryos were transferred to five recipients. The oestrus was prolonged in three recipients and they returned to oestrus on Day 57, 62 and 67 after the previous oestrus. These results indicate that aggregating parthenogenetic embryos can prolong their survival in vitro and in vivo.

Complete preimplantation development in culture of parthenogenetic mouse embryos

Development ◽  
1976 ◽  
Vol 35 (1) ◽  
pp. 179-190
Author(s):  
Matthew H. Kaufman ◽  
Leo Sachs
Keyword(s):  
Polar Body ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Haploid Nucleus ◽  
Second Polar Body ◽  
Haploid Embryos ◽  
Parthenogenetic Mouse Embryos ◽  
Parthenogenetic Embryos ◽  
Better Than

The present experiments were undertaken to determine whether, in parthenogenesis, heterozygous embryos develop better than homozygous embryos. Such experiments may provide an approach to elucidating whether fertilized embryos develop better than parthenogenetic ones because of heterozygosity, or if the sperm provides another contribution necessary for complete embryonic development. The parthenogenetic embryos studied included uniform haploids after extrusion of the second polar body, mosaic haploids in which each blastomere contained a genetically different haploid nucleus, and heterozygous diploid mouse embryos. Eggs were activated and cultured in a chemically denned medium. About three times as many mosaic haploid or heterozygous diploid eggs developed beyond the 4-cell stage after 98–100 h and to the blastocyst stage after 120 h in culture, than uniform haploid eggs. This indicates that the development of parthenogenetic embryos is probably under genetic control and that there was a better development of the heterozygous embryos. Mosaic haploid embryos showed the same high frequency of development as heterozygous diploids. The results therefore indicate that heterozygosity provided a developmental advantage even when distributed between two genetically different clones of cells in the same embryo.

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.

Diploid and haploid mouse parthenogenetic development following in vitro activation and embryo transfer

Development ◽  
1974 ◽  
Vol 31 (3) ◽  
pp. 635-642
Author(s):  
M. H. Kaufman ◽  
R. L. Gardner
Keyword(s):  
Embryo Transfer ◽  
Mouse Embryos ◽  
Parthenogenetic Development ◽  
In Vitro Activation ◽  
Haploid Embryos ◽  
Parthenogenetic Mouse Embryos

Parthenogenetic mouse embryos were selected following in vitro activation, and transferred to the oviducts of pseudopregnant recipients. Decidua was evoked by 50–56% of diploid parthenogenones compared to 35·1% of haploid embryos with a single pronucleus, 37·5% of immediate cleavage eggs and 77% of fertilized eggs (controls). On day 4, 58·7% of diploid parthenogenones were morphologically normal morulae or blastocysts; over 90% of these ‘normal’ embryos evoked decidua when retransferred to recipients compared to 8·9% of abnormal embryos flushed from the ‘transfer’ sides, suggesting that only ‘normal’ embryos could evoke decidua. Potentially diploid parthenogenones remained diploid on chromosomal examination on day 4.

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.

56 THE EFFECT OF VITRIFICATION FOR SHEEP OOCYTES VIABILITY

2015 ◽  
Vol 27 (1) ◽  
pp. 121 ◽  
Author(s):  
Y. M. Toishibekov ◽  
R. K. Tursunova ◽  
M. Sh. Yermekova
Keyword(s):  
Polar Body ◽  
Control Group ◽  
Polar Bodies ◽  
Maturation Medium ◽  
Chi Squared ◽  
Fetal Bovine ◽  
Second Polar Body ◽  
Cryopreservation Techniques ◽  
Humidified Air

Advances in reproduction technologies, such as in vitro maturation, IVF, and in vitro culture, stimulated research for efficient cryopreservation techniques for mammalian oocytes. It is well known that the oocyte is the largest cell of an animal's body and as such, is full of water and, in many species, fat, making it difficult to cryopreserve. The objective of this work was to study the effect of vitrification for cryopreservation of the metaphase II plate (MPII) of sheep oocytes. Ovaries from 20 ewes of Kazakh Arkharo-Merino breed were acquired after slaughter and maintained at 37°C in TCM-199. The maturation medium was TCM-199, containing 1 mM of glutamine, 10% FBS, 5 μg mL–1 FSH, 5 μg mL–1 LH, 1 μg mL–1 oestradiol, 0.3 mM sodium pyruvate, and 100 mM cysteamine. The oocytes were incubated in 400 μL of medium in 4-well dishes covered with mineral oil. The IVM conditions were 5% CO2 in humidified air at 39°C for 24 h. Then they were placed for 10 min in a media with Hoechst 33342 (3 μg mL–1) and cytochalasin B (7 μg mL–1) to facilitate the enucleation of the MPII with a minimum volume of ooplasm. The MPII plates were divided into 2 groups: the vitrification group was exposed to vitrification media containing 1.12 M ethylene glycol (ET) + 0.87 M ME2SO for 5 min and was exposed in vitrification media containing 2.24 M ET + 1.75 M ME2SO for 5 min, and then in vitrification solution containing 4.48 M ET + 40% ME2SO + 0.25 M sucrose for 30 s. Oocytes were loaded into cryoloop and plunged into liquid nitrogen (LN2). Oocytes were thawed in a 25°C water bath and then placed in TCM-199 at 20% fetal bovine serum. After 15 min of incubation the oocytes were activated for extrusion of the second polar body in 1 mg mL–1 Ca ionophore for 5 min and washed for 5 min followed by 4 h in 6-DMAP (0.12 mM) + cycloheximide (0.6 μg mL–1). After activation the MPII were washed and cultured for 20 h. The control group received the same treatment, but they were not vitrified. Differences between the experimental groups were tested using Chi-squared test. Our research showed the expulsion of the second polar body after activation was observed in more than 62.2% of the MPII that were not vitrified (control group), whereas 40.5% of vitrified plates had expulsion of polar bodies (P < 0.05). These preliminary studies showed that it is possible to vitrify MPII plates. On the other hand, the drastic reduction of the volume of the sheep oocytes might make cryopreservation possible with greater efficiency.

Effects of high calcium levels on the disturbed extrusion of the second polar body during in vitro fertilization in C3H/He mouse substrains

Zygote ◽  
2019 ◽  
Vol 28 (1) ◽  
pp. 83-85
Author(s):  
Y. Ohta-Takada ◽  
Y. Nagao ◽  
S. Kito
Keyword(s):  
Calcium Concentration ◽  
Inbred Mice ◽  
Polar Body ◽  
Efficient Production ◽  
Vitro Fertilization ◽  
Practical Applications ◽  
High Calcium ◽  
High Concentrations ◽  
Second Polar Body

SummaryWe previously reported that high concentrations (≥3.42 mM) of calcium during in vitro fertilization (IVF) disturbed the extrusion of the second polar body (PBII) in C3H/He inbred mice. In this study, the substrain specificity of this phenomenon was examined under 1.71–6.84 mM calcium concentration in ova from six C3H/He mouse commercially available substrains in Japan. PBII extrusion in ova from J substrains was not affected by calcium concentrations (<10% at any calcium level), but was grossly disturbed at high calcium levels in the ova of other substrains. This result has practical applications for the efficient production of normal zygotes by IVF, therefore contributing to the reduction in the numbers of donor animals for further zygote or embryo manipulation. Care must be taken in choosing IVF medium for particular strains and substrains.

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