M. L. Semenova

Transfer of foreign DNA into the cells of developing mouse embryos by microprojectile bombardment

Mouse cells of developing embryos at the 2–4 cell, morula and blastocyst stages, were bombarded by high velocity tungsten microprojectiles. About 70% of developing embryos survived the bombardment. The general embryo structure did not change as a result of the bombardment. Penetration of the tungsten microparticles into the embryo cell nuclei was found at all stages being investigated, and tungsten particle localization on mitotic chromosomes was demonstrated. The total DNA of the mice born from the bombarded embryos was analyzed by dot‐blot hybridization and PCR with post‐hybridization. The most important results were obtained in experiments with blastocysts. In three cases of blastocyst bombardment, the presence of transferred plasmid DNA (pSV3‐neo) was revealed. Transfected cells were shown to be located in the fetal membrane as well as in the embryo. The bombardment of mouse culture cells resulted in their transfection and the production of G418‐resistant clones.

Female fertility preservation strategies: cryopreservation and ovarian tissue in vitro culture, current state of the art and future perspectives.

In the present review, the main strategies of female fertility preservation are covered. Procedures of fertility preservation are necessary for women who suffer from diseases whose treatment requires the use of aggressive therapies, such as chemotherapy and radiotherapy. These kinds of therapy negatively influence the health of gametes and their progenitors. The most commonly used method of female fertility preservation is ovarian tissue cryopreservation, followed by the retransplantation of thawed tissue. Another approach to female fertility preservation that has been actively developed lately is the ovarian tissue in vitro culture. The principal methods, advantages and drawbacks of these two strategies are discussed in this article.

Carnosine-induced changes in the development of spermatogenic epithelial cells in senescence accelerated SAM mice.

Carnosine significantly increased the number of spermatogonia and Sertoli cells in mice prone (SAMP1) and resistant (SAMR1) to accelerated aging and appreciably reduced cell yield in meiosis and spermiogenesis in SAMP1 mice. In experimental SAMP1 mice catastrophic changes in the number of gametes were paralleled by intensive degradation of the spermatogenic epithelium. In SAMR1 mice treated with carnosine highly ordered spermatogenic structure was preserved.

Cytogenetic Aberrations in the Cells of Liver and Spermatogenic Epithelium in Senescence Accelerated SAMP1 and SAMR1 Mice

It was shown that during ontogenesis, the mice prone to (SAMP1) and resistant (SAMR1) against accelerates senescence did not differ substantially in the frequency of cytogenetic aberrations in the hepatocytes and spermatogenic cells (spermatogonia and round spermatids). These data suggest that in the mice of both lines, the processes of appearance, development, and functioning of complex biological systems, such as liver and spermatogonic epithelium take place against the background of high genetic instability. The role of genetic instability in senescence is discussed.

Application of femtosecond laser scalpel and optical tweezers for noncontact biopsy of late preimplantation embryos

We demonstrate that one of the key steps of preimplantation genetic diagnosis called embryo biopsy can be successfully performed in noncontact mode by means of femtosecond laser scalpel and optical tweezers. Embryo biopsy was carried out in late-stage mouse preimplantation embryos. Femtosecond laser pulses were applied to detach the desired amount of trophectoderm cells from the blastocyst, while the optical tweezers trapped the cells and moved them out of the embryo. The parameters of laser radiation were optimized so as to efficiently perform embryo biopsy and preserve the viability of the treated embryos.

Application of femtosecond laser pulses in biomedical cell technologies

The results are presented of the works in the field of development of equipment, investigation techniques, and technologies for biology and medicine performed in the Joint Institute for High Temperatures of the Russian Academy of Scienses (JIHT RAS). On the base of the new generation infrared femtosecond lasers, the experimental models are developed and manufactured of laser tweezers, scalpel, and the “tweezers-scalpel” combined system. The results are presented of the experimental studies on the noncontact mammalian cell fusion (blastomeres of mouse embryos on day 1.5 of development) by means of the femtosecond laser pulses.

Biomedical and biotechnology applications of noncontact femtosecond laser microsurgery of living cells

We employed femtosecond (fs) laser pulses to solve important biomedical and biotechnology problems, embryo biopsy and cell fusion respectively. We report on the results of fully contactless laser-mediated polar body (PB) and trophectoderm (TE) biopsy of early mammalian embryos. In the former case the fs laser scalpel (Cr:Forsterite seed oscillator and a regenerative amplifier, 100 fs, 10 Hz) was initially used to drill an opening in the outer covering of the embryo, and then the PB was extracted out of the zygote by means of optical tweezers (cw fiber laser, 1064 nm). In the latter case the laser scalpel was employed to dissect 5-7 TE cells that had just left the zona pellucida (ZP) during the hatching. The energy of laser pulses was thoroughly optimized to prevent cell damage and provide high viability of treated cells. Morphological and fluorescent analysis showed that femtosecond laser-based embryo biopsy did not compromise further in vitro embryo development. We also demonstrated the possibilities of ...

Femtosecond Laser Assisted Hatching: Dependence of Zona Pellucida Drilling Efficiency and Embryo Development on Laser Wavelength and Pulse Energy

Ultrashort laser pulses have enabled highly precise and delicate processing of biological specimens. We present the results of using femtosecond laser pulses for dissection of zona pellucida (ZP) in mouse embryos during assisted hatching procedure. We studied the effects of application of femtosecond laser radiation in the infrared (1028 nm) and visible (514 nm) wavelength ranges. Laser irradiation parameters were optimized so as not to compromise the viability of the treated embryos. We have demonstrated that application of femtosecond laser pulses with the energies in the range of 250–320 nJ (for the wavelength of 1028 nm) and 47–112 nJ (for 514 nm) resulted in efficient ZP dissection. Femtosecond laser-assisted ZP drilling does not slow down the development of pre-implantation embryos and leads to 90–95% frequency of complete hatching. The thermal effects can be significantly lower when femtosecond lasers are used as compared to continuous wave or long-pulse lasers. It is crucial when dealing with living cells or organisms. By optimizing femtosecond laser radiation parameters assisted hatching as well as a wide range of embryo-surgical procedures can be efficiently performed, thus creating a great potential of using femtosecond lasers as a multi-purpose “tool of choice” for specialists in the fields of embryology and developmental biology.

Carnosine modifies the incidence of genetically abnormal sex cells in the testes of senescence-accelerated mice.

The quantitative micronucleus test showed that the natural dipeptide carnosine increases the count of aberrant spermatogonia and round spermatids in the testes in SAMR1 mice resistant to accelerated aging by 64 and 85%, respectively, compared to the control. However, this agent did not modify the incidence of chromosome mutations in spermatogenic cells in SAMP1 mice predisposed to accelerated aging.

Noncontact microsurgery and micromanipulation of living cells with combined system "Femtosecond laser scalpel-optical tweezers"

We report on the results of using self-developed combined laser system consisting of a femtosecond laser scalpel (Cr:Forsterite seed oscillator and a regenerative amplifier, 620 nm, 100 fs, 10 Hz) and optical tweezers (cw laser, 1064 nm) for performing noncontact laser-mediated polar body (PB) and trophectoderm (TE) biopsy of early mammalian embryos. To perform PB biopsy the femtosecond laser scalpel was initially used to drill an opening in the zona pellucida, and then the PB was extracted out of the zygote with the optical tweezers. Unlike PB biopsy, TE biopsy allows diagnosing maternally-derived as well as paternally-derived defects. Moreover, as multiple TE cells can be taken from the embryo, more reliable diagnosis can be done. TE biopsy was performed by applying laser pulses to dissect the desired amount of TE cells that had just left the zona pellucida during the hatching. Optical tweezers were then used to trap and move the dissected TE cells in a prescribed way. Laser power in optical tweezers and energy of femtosecond laser pulses were thoroughly optimized to prevent cell damage and obtain high viability rates. In conclusion, the proposed techniques of laser-based embryo biopsy enable accurate, contamination-free, simple and quick microprocessing of living cells.