The early development of haploid and aneuploid parthenogenetic embryos

Development ◽  
1975 ◽  
Vol 34 (3) ◽  
pp. 645-655
Author(s):  
Matthew H. Kaufman ◽  
Leo Sachs
Keyword(s):  
Early Development ◽  
Polar Body ◽  
Chromosome Counts ◽  
Cell Stage ◽  
Haploid Chromosome Number ◽  
Stage Of Development ◽  
Morula Stage ◽  
Second Polar Body ◽  
Similar Development ◽  
Parthenogenetic Embryos

The early development of parthenogenetically activated oocytes has been studied in C57BL × CBA-T6T6 (F1T6) translocation heterozygote mice and C57BL × CBA-LAC (F1LAC) mice. All F1T6 oocytes had either a quadrivalent or a univalent-trivalent configuration at meiosis I; no such chromosome configurations were observed in the F1LAC oocytes. At ovulation 36·5 % of the F1T6 oocytes had 19 or 21 chromosomes, whereas 97 % of the F1LAC had the normal haploid chromosome number of 20. After parthenogenetic activation, chromosome counts at metaphase of the first cleavage mitosis were made of the eggs with a single pronucleus following extrusion of the second polar body. These activated eggs had similar frequencies of 19, 20 and 21 chromosomes as had the oocytes at ovulation. The activated 1-cell eggs were transferred to the oviducts of pseudopregnant recipients and the embryos recovered 3 days later. At this stage of development, most of the F1T6 embryos with 19 chromosomes were no longer found, but the frequency of 21-chromosome embryos was similar to the frequency of 21-chromosome oocytes and activated eggs. There was a similar mean number of cells in the embryos with 20 and 21 chromosomes. The results indicate that nearly all the embryos with 19 chromosomes failed to develop, probably beyond the 2-cell stage, whereas oocytes with 21 chromosomes had a similar development to oocytes with 20 chromosomes up to the morula stage.

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.

120 EMBRYONIC AND POSTNATAL DEVELOPMENT OF DIPLOID - TRIPLOID MOUSE CHIMAERAS

2005 ◽  
Vol 17 (2) ◽  
pp. 210
Author(s):  
A. Suwinska ◽  
M. Waksmundzka ◽  
W. Ozdzenski ◽  
A.K. Tarkowski
Keyword(s):  
Postnatal Development ◽  
Polar Body ◽  
Experimental Production ◽  
Cell Stage ◽  
Embryonic Tissues ◽  
Glucose Phosphate ◽  
Successful Attempt ◽  
Second Polar Body ◽  
Painting Probes ◽  
Insight Into

A chimaera is an organism composed of cells derived from two (or more) zygotes. Spontaneously originated diploid-triploid (2n-3n) chimaeric embryos and adults have been described in many species of mammals. In man, between 1960 and 2002 over 30 cases of chimaerism were discovered (van de Laar I et al. 2002 Clin. Genet. 62(5), 376–382). A deeper insight into the developmental consequences of this rare and odd phenomenon requires experimental production of 2n-3n embryos and animals. The present study is the first and successful attempt to produce diploid-triploid chimaeric embryos, fetuses, and postnatal animals in the mouse. Diploid embryos originated from BAMIZ females crossed with BAMIZ males. The zygotes that were the source of triploid embryos were obtained from females F1 (C57Bl/6 × CBA/H) crossed with F1 males as a result of “delayed mating.” The triploidy was induced by suppression of the extrusion of the second polar body with cytochalasin D (1 μg mL−1, 5 h). Diploid-triploid chimaeric embryos were created by aggregation of diploid embryos with triploid embryos at 4–8 cell stage. In chimaeras created according to this procedure, the triploid component was agouti and produced the 1B1B isoform of glucose phosphate isomerase (GPI) and the diploid component was albino and produced the GPI-1A1A isoform. Electrophoresis of GPI was performed in order to determine the contribution of both populations of cells in tissues of embryos and individuals. Over a thousand oocytes were subjected to triploidization. A total number of 201 diploid-triploid aggregates developed into blastocysts and were transplanted to the oviducts of 30 recipients. Our experiment yielded 23 living and 6 dead embryos (age: 8th–19th day) out of which 22 proved to be chimaeric and 3 were adults. Two of these animals were albino but had the triploid component in several internal tissues; both were fertile. The third animal, a male, was an overt chimaera. It turned out to be infertile (no sperm in the ejaculate; testes small and deprived of germ cells). The infertility of this individual is puzzling because the FISH studies with the help of X and Y chromosome painting probes proved that the diploid component was XY and the triploid component was XXX. The results of our study indicate that the rate of postimplantation development of 2n-3n chimaeric embryos is normal or only slightly retarded. Developmental stage of chimaeric embryos was assessed by comparison of their external morphology with normal diploid embryos of equivalent post-coital age according to the descriptions given by Theiler K (1972 The House Mouse, Springer-Verlag, Berlin). With the exception of one embryo lacking both eyes (but otherwise looking quite normal) no other morphological abnormalities were observed. Comparison of the contribution of both components to the fetal and extra-embryonic tissues at the consecutive foetal stages has shown that participation of triploid cells slightly but steadily decreased in all tissues examined. However, the presence of triploid cells in mouse chimaeras was compatible with their normal postnatal development to adulthood.

scholarly journals In vitro aging of porcine oocytes

2011 ◽  
Vol 49 (No. 3) ◽  
pp. 93-98 ◽  
Author(s):  
I. Petrová ◽  
M. Sedmíková ◽  
E. Chmelíková ◽  
D. Švestková ◽  
R. Rajmon
Keyword(s):  
In Vitro Culture ◽  
Polar Body ◽  
Cell Stage ◽  
Subsequent Aging ◽  
Parthenogenetic Activation ◽  
In Vitro Aging ◽  
Polar Bodies ◽  
Porcine Oocyte ◽  
Parthenogenetic Embryos

Porcine oocytes matured in vitro develop in various ways if they are further cultivated. In our studies these oocytes were cultivated for 1 to 5 days (in vitro aging). During the 1st day of aging, most of them remained at the stage of metaphase II (98%). Then many oocytes underwent the spontaneous parthenogenetic activation. The portion of activated oocytes reached its peak after 2 or 3 days of aging in vitro (39 or 45%). The portion of fragmented oocytes peaked at the same time (28%). During subsequent aging in vitro (i.e. day 4 or 5 of aging), the portion of lysed oocytes significantly increased (30 or 37%). The highest portion of spontaneously activated parthenogenetic embryos at a pronuclear stage (35%) was observed during the 2nd day of aging in vitro. These pronuclear embryos had mainly one polar body with two pronuclei (47% of all pronuclear embryos) or two polar bodies with one pronucleus (38% of all pronuclear embryos). During the 3rd and 5th day of in vitro aging, there was a significant increase in the portion of parthenogenetic embryos cleaved to the 2-cell or 3-cell stage. When considering the prolonged in vitro culture of porcine oocyte, only the first day of aging should be taken into account, since beyond this time significant changes, i.e. parthenogenesis, fragmentation or lysis, occurred in oocytes under in vitro conditions.  

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.

scholarly journals Increased mitochondrial distribution in early-cleaving embryos indicate successful pre-implantation development

2018 ◽  
Vol 14 (4) ◽  
pp. 512-514
Author(s):  
Nor Shahida Abdul Rahman ◽  
Mimi Sophia Sarbandi ◽  
Wan Hafizah Wan Jusof ◽  
Zolkapli Eshak ◽  
Salina Othman ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Close Association ◽  
Mitochondrial Fission ◽  
Polar Body ◽  
Blastocyst Stage ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Cell Stage ◽  
Second Polar Body

The timing of the first zygotic cleavage is an accurate predictor of embryo quality. Embryos that cleaved early have higher developmental viability compared to their late counterparts. During embryonic development, cleavage is affected by cellular metabolic processes performed by mitochondria and its synergistic interaction with endoplasmic reticulum (ER). However, in depth study on differences of mitochondria and ER ultrastructures in early- cleaving (EC) versus late- cleaving (LC) embryos is limited. This study compares mitochondria and ER ultrastructures of EC versus LC embryos using Confocal Laser Scanning Microscopy (CLSM) and Transmission Electron Microscopy (TEM). Embryos were obtained from female ICR superovulated mice, 28-30 hours post hCG. Two-cell embryos were categorized as early-cleaving (EC), while zygotes with the second polar body and two pronuclei present were categorized as late-cleaving (LC). The LC embryos were cultured in vitro until the 2- cell stage. In EC embryos, mitochondria were mostly found at the perinuclear region and closely associated with dense ER. Meanwhile, mitochondria of LC embryos were distributed uniformly within the cytoplasm. Mitochondrial fluorescence intensity was significantly higher in EC versus LC [(18.7 ± 0.4) versus (14.6 ± 0.4)] x 105 pixel, (p<0.01). Development to the blastocyst stage was also significantly higher in EC compared to LC embryos (96.7% versus 60.9%) (p<0.01). Higher viability of EC embryos is attributed to the close association of their mitochondria to ER. This contributed to better mitochondrial fission, resulting in enhanced energy generating processes and preimplantation development. 

scholarly journals 271 ASSESSMENT OF NUCLEAR STATUS OF ACTIVATED BOVINE OOCYTES MATURED IN DIFFERENT MATURATION CONDITIONS IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 285
Author(s):  
J.I. Park ◽  
Y. Jang
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Calf Serum ◽  
Chi Square ◽  
Cell Stage ◽  
Maturation Medium ◽  
Second Polar Body ◽  
Humidified Air ◽  
Bovine Oocytes

This study was carried out to assess the nuclear status after parthenogenetic activation in in vitro matured oocytes under different conditions. Bovine ovaries were collected from slaughtered cows at a local abattoir. Oocytes were aspirated from follicles of 3–8 mm in diameter and transferred to maturation medium: tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal calf serum, 100 mg/mL l-cysteine, 20 mg/mL sodium pyruvate, gonadotropins (each 250 IU of eCG and hCG/mL), and 10 mg/mL epidermal growth factor, with or without 5 mM hypotaurine and taurine. Oocytes were cultured at 38.9°C in 5% CO2 in humidified air. After 24 h of culture, oocytes with polar body were selected and submitted to activation treatments. Oocytes were exposed to calcium ionomycin (5 μM for 5 min) followed by incubation with 6-DMAP (2 mM), roscovitine (50 μM), or 6-DMAP + roscovitine for 3.5 h. After activation, oocytes were cultured in mSOF medium containing 0.8% BSA at 38.9°C in 5% CO2, 5% O2 in humidified air for 16 h and stained with Hoechst 33342 or aceto-orcein for assessment of nuclear status. Nuclear status was recorded as follows: 1PB (polar body) + 1PN (pronucleus), 2PB + 1PN and others. Data were analyzed using chi-square test. The maturation rate of bovine oocytes cultured in maturation medium containing hypotaurine/taurine (89.3%, n = 84) was higher (P < 0.05) than those cultured without hypotaurine/taurine (72%, n = 93). In the oocytes matured with hypotaurine/taurine, the rates of diploid activation (1PB + 1PN) were 84% (n = 50) in oocytes treated with 6-DMAP + roscovitine, 78.6% (n = 56) with 6-DMAP, and 52% (n = 50) with roscovitine. In the oocytes matured without hypotaurine/taurine, the rates of diploid activation were 80% (n = 60) in oocytes treated with 6-DMAP + roscovitine, 72% (n = 50) with 6-DMAP, and 54% (n = 50) with roscovitine. The rates of diploid activation were not different in oocytes matured with or without hypotaurine/taurine and among activation treatments. The oocytes treated with roscovitine showed a lower rate (P < 0.05) of diploid activation and higher rate (39.3–40%) of second polar body extrusion (1PN + 2PB) than the other activation groups in both maturation conditions. Cleavage rates to 2-cell stage were 40–45% in all groups. Development rate of blastocysts were 7–10% in all the groups treated with 6-DMAP and 6-DMAP + roscovitine and no blastocysts were obtained from the groups treated with roscovitine alone. Hypotaurine/taurine are known to be stable and potent antioxidants, and have shown the properties of supporting oocyte maturation and further embryonic development (Guerin and Menezo 1995 Zygote 3, 333–43; Mizushima and Fukui 2001 Theriogenology 55, 1432–45). In this study, although the effectiveness of hypotaurine/taurine on promoting oocyte maturation was observed, there were no significant improvements in the rate of diploid activation in oocytes matured with hypotaurine/taurine. These results suggest that the nuclear status of activated oocytes may not have a direct relationship with the enhanced maturation condition. This work was supported by BioGreen 21 Program(#1000520030100000-1), Republic of Korea.

211 IN VITRO PRODUCTION OF SWAMP BUFFALO EMBRYOS BY INTRACYTOPLASMIC SPERM INJECTION: EFFECT OF CHEMICAL ACTIVATION TREATMENTS

2009 ◽  
Vol 21 (1) ◽  
pp. 203
Author(s):  
Y. Y. Liang ◽  
D. N. Ye ◽  
C. Laowtammathron ◽  
T. Phermthai ◽  
R. Parnpai
Keyword(s):  
Polar Body ◽  
Chemical Activation ◽  
Cumulus Cells ◽  
Success Rates ◽  
In Vitro Production ◽  
Cell Stage ◽  
Sham Injection ◽  
Swamp Buffalo ◽  
Second Polar Body

Intracytoplasmic spern injection (ICSI) in the buffalo has not yet been well examined. Several factors involved affect the success rates of this technique, particularly the postinjection activation procedure. The objective of this study was to evaluate the effects of chemical activation treatments on in vitro development of oocytes after ICSI. A single spermatozoa was injected into the cytoplasm of an in vitro-matured oocyte using a micromanipulator under an inverted microscope. The ICSI oocytes were assigned to the following chemical activation treatments: (1) exposed to 5 μm ionomycin (Io) in Emcare medium for 5 min and placed in Emcare medium for 3 h, or (2) exposed to 7% ethanol (EtOH) in Emcare medium for 5 min and placed in Emcare medium for 3 h. The treated oocytes that extruded a second polar body were then selected and cultured either in (A) 1.9 mm 6-dimethylaminopurine (6-DMAP) in mSOF medium for 3 h, or (B) 10 μg mL–1 of cychloheximide (CHX) for 5 h. The treated oocytes were further cultured in mSOF medium supplemented with 3 mg mL–1 of fatty acid-free BSA at 38.5°C under a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 for 2 d. Thereafter, 8-cell-stage embryos were selected and co-cultured with buffalo cumulus cells in mSOF medium at 38.5°C under a humidified atmosphere of 5% CO2 in air for another 5 d. The medium was changed daily and the development of embryos was recorded at the same time the medium was changed. The sham-injected oocytes were treated and cultured along with ICSI oocytes. With 8 replications for each activation treatment, 336 oocytes were used for ICSI. With 6 replications for each activation treatment, 211 oocytes were used for sham injection. The cleavage of ICSI oocytes treated with Io + 6-DMAP, EtOH + 6-DMAP, and EtOH + CHX was 76.2, 69.4, and 78.3%, respectively, which was significant higher (P < 0.01) than ICSI oocytes treated with Io + CHX (52.4%) and also significant higher (P < 0.01) than sham-injected oocytes in all treatments. The highest blastocyst rate was observed in ICSI oocytes treated with Io + 6-DMAP (28.6%), which was not significantly different from ICSI oocytes treated with EtOH + CHX (24.4%). The blastocyst rates of ICSI oocytes treated with Io + 6-DMAP and EtOH + CHX were significantly higher than ICSI oocytes treated with Io + CHX (5.9%) and EtOH + 6-DMAP (16.5%) and also were significantly higher than sham-injected oocytes in all treatments. In conclusion, our study demonstrated that activated ICSI of swamp buffalo oocytes with Io + 6-DMAP or EtOH + CHX gave the highest cleavage and blastocyst rates. This work was supported by the Thailand Research Fund and Suranaree University of Technology.

Spontaneous Activation of the Hamster Egg in vivo

1973 ◽  
Vol 31 ◽  
pp. 548-549
Author(s):  
F. J. Longo
Keyword(s):  
Polar Body ◽  
Light And Electron Microscopy ◽  
Microscopic Level ◽  
Estrus Cycle ◽  
Light Microscopic Level ◽  
Cell Stage ◽  
Spontaneous Activation ◽  
Second Polar Body ◽  
Fertilized Egg

Spontaneous activation of the hamster ovum is a normal occurence in oviducal eggs that are not inseminated. Many aspects comprising this process of parthenogenesis mimic events characteristic of fertilization and include for example, the formation of the second polar body and the development of one or two pronuclei. Occasionally the activated egg cleaves to form a two-cell stage. The similarity between the spontaneously activated hamster ovum and the inseminated egg has been previously documented at the light microscopic level of investigation; however, further investigation is warranted for the study of parthenogenesis in mammals provides an opportunity of elucidating developmental mechanisms of the fertilized egg. Accordingly, unfertilized eggs were flushed from the oviducts of hamsters at different intervals during the estrus cycle (6 to 70 hours postovulation), prepared for light and electron microscopy, and compared with fertilized eggs obtained at corresponding periods.

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.

Comparative expression analysis of embryonic development-related genes at different stages of parthenogenetic and in vitro fertilized embryos in caprine

Zygote ◽  
2013 ◽  
Vol 23 (2) ◽  
pp. 198-204 ◽  
Author(s):  
Renu Singh ◽  
Kuldeep Kumar ◽  
R. Ranjan ◽  
Manish Kumar ◽  
T. Yasotha ◽  
...  
Keyword(s):  
Gene Expression ◽  
Developmental Competence ◽  
Cell Stage ◽  
Developmental Abnormalities ◽  
Aberrant Gene Expression ◽  
Comparative Expression Analysis ◽  
Morula Stage ◽  
Aberrant Gene ◽  
Parthenogenetic Embryos

SummaryAberrant gene expression occurs in parthenogenetic embryos due to abnormal epigenetic modifications in the genome that probably diminish viability and enhance developmental abnormalities in these embryos. In the present study, five developmentally important genes (HPRT1, Cx43, Sox2, Mest and IGF2R) were analysed at different stages in parthenotes (haploid and diploid) and compared with similar stages in in vitro fertilized (IVF) embryos. The results indicated that in haploid parthenotes expression of HPRT1 was upregulated (P < 0.05) only at the 2–4-cell stage whereas Cx43 expression was significantly (P < 0.05) downregulated in all stages as compared with the control. However, expression of this gene was upregulated (P < 0.05) in 2–4-cell and morula stages of diploid parthenotes. Expression of Sox2 was significantly (P < 0.05) downregulated in morula stage haploid parthenotes, whereas it was upregulated (P < 0.05) in 8–16-cell stage diploid embryos. The expression of Mest was upregulated (P < 0.05) at the 2–4-cell stage of both haploid and diploid parthenotes, whereas it was downregulated in 8–16-cell stage diploid embryos as compared with control. IGF2R expression was upregulated (P < 0.05) only in morula stage haploid and diploid parthenote as compared with control. These results indicate that parthenogenetic embryos showed aberrant gene expression of developmentally important genes such as HPRT1, Cx43, Sox2, Mest and IGF2R in comparison with IVF embryos, this finding may be one of the major reasons for the poor developmental competence of parthenogenetic embryos.

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