In vitro fertilization techniques with isolated angiosperm gametes have been developed recently. Zygotes, embryos and fertile plants can now be obtained from. The rice in vitro fertilization (IVF) system is a suitable system to elucidate the relationship between the gamete fusion point on the zygote and. This new single cell, called a zygote, contains all of the genetic material needed to form a As you can see, the first sperm to reach the oocyte is never the one to fertilize it. The ideal ratio is 75, sperm to one egg. In vitro fertilization involves egg collection from the ovaries, fertilization in a petri dish, and the transfer of.
Moreover, using free-living gametes of brown algae, it has been revealed that the sperm entry site functions as positional information for establishing an intermediate default axis of the zygote, which is then overridden by light stimuli that cause the formation of the thallus—rhizoid axis Kropf, ; Hable and Kropf, These reports suggest that the gamete fusion activates the egg cell, and that the gamete fusion site marks the apical—basal axis orientation of the zygote and proembryo.
The rice in vitro fertilization IVF system is a suitable system to elucidate the relationship between the gamete fusion point on the zygote and the position of the cleavage plane in the two-celled proembryo, since the IVF-produced rice zygote divides into an asymmetric two-celled proembryo consisting of a small apical cell with dense cytoplasm and a large basal cell with well-developed vacuoles in a highly similar manner to the zygote within the embryo sac Uchiumi et al.
However, since the gamete fusion site cannot be traced using the conventional IVF system, the IVF system requires modification to enable labelling of the gamete fusion site and for subsequent tracing of the fusion site in relation to the first division plane of the zygote. In the present study, the gamete fusion site on the rice zygote was fluorescence-labelled, and the positional relationship between the gamete fusion site and the zygotic first division plane was monitored to judge whether the gamete fusion point functions as a putative positional cue for the positioning of the zygote cleavage plane.
Isolation of rice egg and sperm cells and electrofusion-mediated IVF using the isolated gametes were conducted as described by Uchiumi et al. Culture of zygotes Two methods were used for zygote culture.
One was a Millicell-based culture Uchiumi et al. These dishes were placed in mm-diameter plastic dishes filled with 2 ml of N6Z medium Kumlehn et al. Then, 5—10 cell aggregates of cultured cells were transferred from the cultured dishes into a culture droplet, as mentioned above, under an inverted microscope Olympus BX, Tokyo, Japan. After incubation for 1—2 min, cells were washed by transferring them into fresh mannitol droplets three times and a fluorescent-labelled sperm cell was fused with an egg cell as described above.
In a Millicell or droplet, one to three zygotes were cultured. Fluorescent observation of the sperm cells or the fusion point on the zygote was conducted using a BX inverted fluorescence microscope with — nm excitation and — nm emission wavelengths U-MWIBA2 mirror unit; Olympus.
Results Fluorescent labelling of the gamete fusion site on the rice zygote The plasma membrane of isolated sperm cells was stained with concanavalin A conjugated with Alexa Fluor ConA as described in the Materials and methods Fig.
In IVF practice, hazardous manipulation may lead to such anomalies. It is very difficult to estimate the quality of oocytes at the time of ovum retrieval as there is no correlation between cumulus maturation and true oocyte quality. Using classical techniques, an interpretable karyotype was obtained from These oocytes were classified according to a cytological criterion of maturity presence or absence of the first polar body ; 39 oocytes displaying no polar body were judged immature.
Chromosome analysis showed 8 oocytes in diakinesis, and 31 metaphase-II diploid oocytes, including 10 presenting prematurely condensed paternal chromosomes into single chromatids PCC. Among the oocytes having extruded the first polar body, and presumably in metaphase-II, Mainly small chromosomes were involved.
Sperm chromosomes presenting single chromatids were present in These studies point out a higher rate of hyperhaploidy in the oocytes in metaphase-II. These findings have been confirmed in our own research.In Vitro Fertilization (IVF) Video - Brigham and Women's Hospital
Our observations of the first group of 39 oocytes also corroborated these results and demonstrated the possibility of metaphase-II diploidy without extrusion of the first polar body, and the more frequent occurrence of hyperhaploidy for small chromosomes which are more exposed to non-disjunction.
The results of this last study and our data are inconsistent with the correlation reported between maternal age and the rate of abnormalities. Our two techniques of stimulation resulted in a similar rate of aneuploidy, and did not affect the pre-existing chromosome equilibrium of the oocyte.
Cytogenetic analysis of these human eggs is a useful tool to assess the rate of aneuploidy at different stages of female meiosis.
Embryos General aspect The two most useful predictors of viability of preimplantation embryos are i normal morphology, and ii fast cleavage rate. As there are often faults in assessing these morphological parameters, a good understanding of metabolism becomes an additional prerequisite for any test of embryo quality.
Theoretically, any molecule incorporated by the embryo may serve as a basis for a quality test. Because of the size of the embryo, this approach is not that easy. The problem is then to develop a sufficiently sensitive system to quantify the transport of these molecules between the egg and its culture medium generally by using radioisotopes.
For human eggs no tests are presently available, as the use of radioisotopes is not possible. In animals, several experiments have demonstrated that hexose uptake is useful to determine embryo quality in farm animals 17 Table 1.
Another approach is the study of chemical messages released by the egg early pregnancy factors [EPF], cytokins and other luteotrophic factors.
These specific factors influence ovum transport and ovarian activity in vivo. To increase embryonic quality in vitro, several coculture systems have recently been designed These systems allow a selection in vitro of the best embryos. It is not easy to determine the future of the embryo in vivo as genital tract secretions may interfere positively, but also negatively with embryo development and implantation.
FISH Early stage embryos. Non transferable, non freezable embryos with four to eight blastomeres presenting morphological or cytological abnormalities were elected from IVF programs.
Gamete and embryo quality
A culture with colcemid was performed during 7 hours. Chromosome preparations were obtained using a variant of the air drying method of Tarkowsky FISH with X probes displayed 21 male and 26 female embryos. FISH with 9 and 18 probes revealed one 9 trisomy, one 18 trisomy and one 9 monosomy.
This study confirmed that the development of zygotes may be interrupted at different stages of the first somatic division. This event may be correlated with cytoplasmic immaturity 1. The biotin labelled DNA probes give significant chromosome hybridization signals in metaphase and interphase nuclei with all immunological systems used, both with phase contrast and fluorescence microscopy. In early embryos, we observed aneuploidies.
We recently demonstrated 4that mixoploidy appears to be a normal feature in preimplantation embryos and that it occurs very early in human embryo development at least at the morula blastocyst transition.
Conclusion A viable embryo must pass harmoniously through several critical steps: The oviduct environment may sometimes have to compensate for certain temporary deficiencies of the new genome; the uterine regulations both positive and negative allow growth towards implantation.