Femtosecond laser-induced blastomere fusion results in embryo tetraploidy by common metaphase plate formation.

The cell fusion is a widespread process, which takes place in many systems in vivo and in vitro. Fusion of cells is frequently related to tetraploidy, which can be found within natural physiological conditions, e.g., placentation, and in pathophysiological conditions, such as cancer and early pregnancy failure in humans. Here we investigate the mechanism of tetraploidization with help of femtosecond laser-induced mouse blastomere fusion by the means of Hoechst staining, GFP, BODIPY dyes and fluorescent species generated intracellularly by a femtosecond laser. We establish diffusive mixing of cytosol, whereas the large components of a cytoplasm (organelles, cytoskeleton) are poorly diffusible and are not completely mixed after cell fusion and a subsequent division. We show that mechanisms which are responsible for the formation of a common metaphase plate triggered tetraploidization in fused mouse embryos and could be a significant factor in polyploidy formation in vivo. Thus, our results suggest that microtubules play a critical role in tetraploidization.

Effect of laser optoperforation of the zona pellucida on mouse embryo development in vitro.

The effect of laser optical perforation of the zona pellucida on the viability and development of mouse embryos has been studied. Operations of zona pellucida thinning and single or double perforation were carried out on 2-cell embryo, morula, and blastocyst stages with a laser pulse (wavelength 1.48 µm, pulse duration 2 ms). Embryo development up to the blastocyst stage and hatching efficiency were statistically analyzed. It was found that 2-cell or morula stage embryo zona pellucida thinning or single perforation did not affect development to the blastocyst stage and number of hatched embryos, but it accelerated embryo hatching compared to control groups one day earlier in vitro. Double optoperforation on 2-cell embryo or morula stage did not significantly affect development to the blastocyst stage, but it strongly decreased the number of hatched embryos. Also, zona pellucida perforation at the blastocyst stage had a negative effect: hatching did not occur after this manipulation. Blastocyst cell number calculation after single zona pellucida perforation at 2-cell and morula stages showed that cell number of hatching or hatched blastocysts did not differ from the same control groups. This fact points out that the laser single optoperforation method is a useful and safe experimental tool that allows further manipulations within the zona pellucida.

Multi-Pulse laser ablation modeling with applications to automated zona removal.

Laser zona drilling (LZD), the ablation of a portion of the zona pellucida (ZP) in embryos with the use of a laser, is a required step in many embryonic surgical procedures such as assisted hatching and preimplantation genetic diagnosis. The objective of LZD is to remove specific locations of the ZP while minimizing potential harmful thermal effects to important structures of the embryo, namely the blastomeres. Current thermal analyzes of lasers used in LZD only encompass the use of a single pulse, whereas LZD is typically performed using multiple pulses. In this paper we analyze the effect of multipulse LZD and introduce a linear approximation method for multi-pulse LZD. Furthermore, we describe a novel method of measuring the thermal effect of a single laser pulse using the thermosensitive fluorescent dye Rhodamine B and a high speed camera.

Manipulating and imaging the early Xenopus laevis embryo.

Over the past half century, the Xenopus laevis embryo has become a popular model system for studying vertebrate early development at molecular, cellular, and multicellular levels. The year-round availability of easily fertilized eggs, the embryo's large size and rapid development, and the hardiness of both adults and offspring against a wide range of laboratory conditions provide unmatched advantages for a variety of approaches, particularly "cutting and pasting" experiments, to explore embryogenesis. There is, however, a common perception that the Xenopus embryo is intractable for microscope work, due to its store of large, refractile yolk platelets and abundant cortical pigmentation. This chapter presents easily adapted protocols to surmount, and in some cases take advantage of, these optical properties to facilitate live-cell microscopic analysis of commonly used experimental manipulations of early Xenopus embryos.

Laser killing of blastomeres in Caenorhabditis elegans.

The Caenorhabditis elegans embryo is particularly amenable to microscopy and embryological studies because of its short developmental time, transparent shell, and nonpigmented cells. Within the embryo, contacts between cells often establish the polarization of neighboring cells. Blastomere isolation and recombination experiments have led to a wealth of understanding of the events in the four-cell C. elegans embryo. However, identifying individual blastomeres after isolation at stages past the four-cell stage is limited. In addition, removal of blastomeres from their native surroundings can interfere with many cell contacts besides the contacts of interest. An alternative approach for studying cell interactions within the C. elegans embryo is to use laser ablation of individual cells. Laser ablation can be used to kill one of two cells in contact with each other to understand what happens when a cell no longer signals to its neighbor. Additionally, killing a cell that is between two cells that will eventually contact each other can result in the corpse of the cell forming a steric barrier between the cells, preventing the contact. This protocol describes laser ablation of embryos mounted on an agar mount.

[Noninvasive optical laser technologies for the transplantation of mammalian nuclei].

The results of pioneering studies on the development of radically new noninvasive methods for the transplantation of mammalian somatic cell nuclei with the use of optical laser manipulations are presented, and their comparison with traditional invasive methods is performed. It is shown that all the key steps, including the enucleation of a recipient cell, the transfer of a somatic cell (karyoplast), its bringing close together with the recipient cytoplast, and the fusion of the cytoplast with the somatic cell, can be effectively conducted using the laser only with the complete replacement by laser of traditional mechanical micromanipulators and other devices, including devices for electrofusion. The results indicate the unique potentialities of laser and the prospects of its application in modern cell engineering in a wide spectrum of studies on oocytes and early mammalian embryos, in particular in technologies of therapeutic and reproductive cloning.

Caenorhabditis elegans APN-1 plays a vital role in maintaining genome stability.

We previously showed that Caenorhabditis elegans APN-1, the only metazoan apurinic/apyrimidinc (AP) endonuclease belonging to the endonuclease IV family, can functionally rescue the DNA repair defects of Saccharomyces cerevisiae mutants completely lacking AP endonuclease/3'-diesterase activities. While this complementation study provided the first evidence that APN-1 possesses the ability to act on DNA lesions that are processed by AP endonucleases/3'-diesterase activities, no former studies were conducted to examine its biological importance in vivo. Herein, we show that C. elegans knockdown for apn-1 by RNAi displayed phenotypes that are directly linked with a defect in maintaining the integrity of the genome. apn-1(RNAi) animals exhibited a 5-fold increase in the frequency of mutations at a gfp-lacZ reporter and showed sensitivities to DNA damaging agents such as methyl methane sulfonate and hydrogen peroxide that produce AP site lesions and strand breaks with blocked 3'-ends. The apn-1(RNAi) worms also displayed a delay in the division of the P1 blastomere, a defect that is consistent with the accumulation of unrepaired lesions. Longevity was only compromised, if the apn-1(RNAi) animals were challenged with the DNA damaging agents. We showed that apn-1(RNAi) knockdown suppressed formation of apoptotic corpses in the germline caused by an overburden of AP sites generated from uracil DNA glycosylase mediated removal of misincorporated uracil. Finally, we showed that depletion of APN-1 by RNAi partially rescued the lethality resulting from uracil misincorporation, suggesting that APN-1 is an important AP endonuclease for repair of misincorporated uracil.

Progress toward the culture and transformation of chicken blastodermal cells.

Chicken blastodermal cells can be cultured for short periods of time and retain the ability to contribute to somatic and germline tissues when injected into gamma-irradiated stage X embryos. Such a method has yet to yield a germline transgenic bird, in part due to the low rate of transgene integration into the avian genome. In addition, the short culture period precludes the identification and expansion of those cells that carry an integrated transgene. In this study, two methods were developed that produced blastodermal cells isolated from stage X Barred Plymouth Rock embryos bearing an integrated transgene. Addition of chick embryo extract to the culture medium enabled expansion of single colonies for multiple passages. Southern blot analysis indicated that the transgenes had integrated as a single copy in most of the clones. Cells from passaged, transgenic embryo cells were injected into irradiated stage X White Leghorn embryos, producing hatched chicks that bore the donor cells in their somatic tissues. Transgene sequences were detected in sperm DNA; however, breeding of chimeras did not result in germline transmission of the transgene, indicating that the contribution of the transgenic cells to the germline was either nonexistent or very low.

Ultraviolet irradiation of eggs and blastomere isolation experiments suggest that gastrulation in the direct developing ascidian, Molgula pacifica, requires localized cytoplasmic determinants in the egg and cell signaling beginning at the two-cell stage.

Gastrulation in the maximum direct developing ascidian Molgula pacifica is highly modified compared with commonly studied "model" ascidians in that endoderm cells situated in the vegetal pole region do not undergo typical invagination and due to the absence of a typical blastopore the involution of mesoderm cells is highly modified. At the gastrula stage, embryos are comprised of a central cluster of large yolky cells that are surrounded by a single layer of ectoderm cells in which there is only a slight indication of an inward movement of cells at the vegetal pole. As a consequence, these embryos do not form an archenteron. In the present study, ultraviolet (UV) irradiation of fertilized eggs tested the possibility that cortical cytoplasmic factors are required for gastrulation, and blastomere isolation experiments tested the possibility that cell signaling beginning at the two-cell stage may be required for the development of the gastrula. Irradiation of unoriented fertilized eggs with UV light resulted in late cleavage stage embryos that failed to undergo gastrulation. When blastomeres were isolated from two-cell embryos, they developed into late cleavage stage embryos; however, they did not undergo gastrulation and subsequently develop into juveniles. These results suggest that cytoplasmic factors required for gastrulation are localized in the egg cortex, but in contrast to previously studied indirect developers, these factors are not exclusively localized in the vegetal pole region at the first stage of ooplasmic segregation. Furthermore, the inability of embryos derived from blastomeres isolated at the two-cell stage to undergo gastrulation and develop into juveniles suggests that important cell signaling begins as early as the two-cell stage in M. pacifica. These results are discussed in terms of the evolution of maximum direct development in ascidians.

Comparison of the results of human embryo biopsy and outcome of PGD after zona drilling using acid Tyrode medium or a laser.

BACKGROUND: Zona pellucida opening for blastomere removal can be done by mechanical or chemical means, or by laser. So far, only limited data on the use of laser systems for zona drilling in cases of PGD are available. METHODS: Results of embryo biopsy and outcome of PGD in two periods were compared. In the first period, acid Tyrode medium was used for zona drilling. In the second period, zona drilling was performed by a 1.48 micro m infrared laser. RESULTS: In the first period, 59 cycles resulted in 53 biopsy procedures with 356 biopsied embryos. In the second period, these numbers were 69 cycles, 69 biopsy procedures and 402 biopsied embryos. Fewer blastomeres were intact (95.2%) after zona drilling with acid Tyrode than after laser zona drilling (98.3%, P = 0.02). Rates of positive HCG (37.5% versus 35.5%), ongoing pregnancy rates (31.3% versus 25.0%) and ongoing implantation rates (18.9% versus 14.9%) did not differ. CONCLUSIONS: The use of a laser for zona drilling in cases of PGD is an easier procedure and results in more intact blastomeres. Since similar pregnancy rates are obtained, it is advantageous to use a laser for zona drilling. Further follow-up is necessary to prove the safety of this procedure.

Laser-assisted removal of necrotic blastomeres from cryopreserved embryos that were partially damaged.

OBJECTIVE: To examine whether the developmental potential of embryos that were partially damaged after freezing and thawing can be improved by removal of necrotic blastomeres before embryo transfer. DESIGN: Prospective pilot study and observational clinical series. SETTING: Private hospital. PATIENT(S): Two hundred thirty-five infertile couples undergoing frozen embryo transfer. INTERVENTION(S): Removal of necrotic blastomeres from frozen-thawed human embryos. MAIN OUTCOME MEASURE(S): Pregnancy and implantation rates. RESULT(S): Removal of necrotic blastomeres from partially damaged frozen-thawed embryos before transfer increased rates of pregnancy (45.7% vs. 17.1%), ongoing pregnancy (40.0% vs. 11.4%) and ongoing implantation (16.2% vs. 4.3%) compared with the control group, in which necrotic blastomeres were not removed. A similarly high implantation rate (16.7%) was seen a subsequent clinical series in which necrotic blastomeres were removed from all partially damaged embryos. CONCLUSION(S): The viability of partially damaged frozen-thawed embryos can be improved by removal of necrotic blastomeres before embryo transfer.

[The simulation of the cooperative effect of development in a culture of early mouse embryos after irradiation with electromagnetic waves in the millimeter range]. / Imitatsiia kooperativnogo éffekta razvitiia v kul'ture rannikh zarodyshei myshi posle oblucheniia élektromagnitnymi volnami millimetrovogo diapazona.

We have found that two-cell mouse embryos cultured in vitro can be stimulated by electromagnetic irradiation in the millimeter range. After 30 min of exposure, they acquire the ability to develop in culture on their own and can reach the stage of blastocyst in a relatively large volume of Whitten cultural medium (150 microliters) without serum or growth factors. It is proposed that millimeter range electromagnetic waves activate metabolic processes and specifically the synthesis of factors controlling early embryonic development in culture.

Dorsoventral polarization and formation of dorsal axial structures in Xenopus laevis: analyses using UV irradiation of the full-grown oocyte and after fertilization.

Xenopus laevis embryos which had been UV-irradiated as full-grown oocytes (UV-O) or after fertilization (UV-F) showed typical UV syndrome, namely dorsal axial deficiency. Morphological comparison revealed that UV-O embryos showed a clear dorsoventral polarity from early cleavage to gastrula stage, but UV-F embryos showed radially symmetrical development throughout embryogenesis. Although UV-O embryos developed morphologically normal-looking dorsal lips of the blastopore, they failed to develop dorsal axial structures at later stages. Implantation of dorsal lips demonstrated that the dorsal lip of UV-O embryos had less activity as Spemann's organizer than the dorsal lip of normal embryos. It is thus suggested that a morphological differentiation of the dorsal lip of the blastopore does not necessarily imply a functional differentiation of Spemann's organizer. Dorsal or ventral cytoplasm from normal embryos at the 8-16 cell stage was injected into a blastomere of UV-F and UV-O embryos at the same stage as the donor. The injection of the dorsal cytoplasm could rescue partially the UV syndrome of UV-F but not of UV-O embryos.

Microinjection of FITC-dextran into mouse blastomeres to assess topical effects of zona photoablation.

The objective of the current experiments was to investigate whether all or only some blastomeres from precompacted mouse embryos were affected by zona photoablation. The microbeam of xenon chloride excimer laser (308 nm) was guided through an inverted microscope (non-contact system). Topical effects of lasing were determined by microinjection of a vital fluorescent dye of high molecular weight (fluorescein isothiocyanate [FITC] dextran) into the cell immediately adjacent to the site of zona photoablation. This dye is only passed onto daughter blastomeres and therefore allows study of specific cell lines. Embryonic growth was assessed following cell separation at the morula and blastocyst stage. Four-cell embryos treated with the laser had significantly fewer cells 12 h after zona photoablation than control embryos. A similar effect was noted after 24 h between dye injected embryos and those injected and lased simultaneously, indicating potential toxic effects of the laser treatment on the embryo. Effects on the blastomere closest to the site of ablation were evaluated by calculating the ratio of dyed cells to the total number of cells at specific time intervals. The ratios were similar in the dye and laser+dye groups of treated 4-cell embryos 36 h after treatment (0.22 and 0.23, respectively), indicating that the dye was still present in approximately 25% of the cells and that the negative effect of photoablation was evenly distributed among the blastomeres. It is concluded that zona photoablation may have long-term detrimental effects of a non-topical nature on precompacted mouse embryos in spite of the apparent precision of the laser spot size.

Cellular interactions involved in the determination of the early C. elegans embryo.

Classical work implied that early nematode embryogenesis is completely mosaic. This view was lately challenged by the demonstration that in C. elegans an early interaction has to occur to induce the production of muscle from a blastomere. Here, early embryonic blastomeres were inactivated by laser microsurgery. The cell lineages of irradiated embryos were compared to those of intact embryos. It is shown that one blastomere, MS, is required for the specification of mesodermal pharyngeal fates and another blastomere, P2, for the specification of hypodermal fates from the descendants of the AB blastomere, whereas the proper specification of the nervous system requires the presence of both. The irradiation of a third blastomere shows that interactions also occur within the ectoderm. I propose that the body plan of the C. elegans embryo may be established by two primary signals followed by secondary interactions. The suggested mechanisms are reminiscent of those involved in amphibian development.

Centrosome movement in the early divisions of Caenorhabditis elegans: a cortical site determining centrosome position.

In Caenorhabditis elegans embryos, early blastomeres of the P cell lineage divide successively on the same axis. This axis is a consequence of the specific rotational movement of the pair of centrosomes and nucleus (Hyman, A. A., and J. G. White. 1987. J. Cell Biol. 105:2123-2135). A laser has been used to perturb the centrosome movements that determine the pattern of early embryonic divisions. The results support a previously proposed model in which a centrosome rotates towards its correct position by shortening of connections, possibly microtubules, between a centrosome and a defined site on the cortex of the embryo.

Radiosensitivity variation during the cell cycle in pronuclear mouse embryos in vitro.

The radiosensitivity of pronuclear mouse (B6D2 F2 X ICR) embyros has been measured in vitro as a function of time during the cell cycle. This was done by measuring the dose of X-rays (LD50) required to prevent development of 50% of the pronuclear embryos to the blastocyst stage in 5 days of culture. The LD50 was found to vary from 1 to 2 Gy during the period from G1 to the first cleavage. The cell cycle in the pronuclear embryo was analysed by [3H]thymidine autoradiography. Compared with earlier studies on two-cell mouse embryo radiosensitivity, the pronuclear embryos appear to be more sensitive to radiation than the two-cell embryos. If, however, one considers the radiation sensitivity on a blastomere basis, the pronuclear embryos are not different in their radiation sensitivity from the two-cell embryos. Thus, during the early cleavage stages of mice, radiosensitivity is mainly governed by the content of cells of various cell cycle ages in the embryo.