Importin α7 Is Essential for Zygotic Genome Activation and Early Mouse Development

Importin α is involved in the nuclear import of proteins. It also contributes to spindle assembly and nuclear membrane formation, however, the underlying mechanisms are poorly understood. Here, we studied the function of importin α7 by gene targeting in mice and show that it is essential for early embryonic development. Embryos lacking importin α7 display a reduced ability for the first cleavage and arrest completely at the two-cell stage. We show that the zygotic genome activation is severely disturbed in these embryos. Our findings indicate that importin α7 is a new member of the small group of maternal effect genes

Biopsy of human morula-stage embryos: outcome of 215 IVF/ICSI cycles with PGS.

Preimplantation genetic diagnosis (PGD) is commonly performed on biopsies from 6-8-cell-stage embryos or blastocyst trophectoderm obtained on day 3 or 5, respectively. Day 4 human embryos at the morula stage were successfully biopsied. Biopsy was performed on 709 morulae from 215 ICSI cycles with preimplantation genetic screening (PGS), and 3-7 cells were obtained from each embryo. The most common vital aneuploidies (chromosomes X/Y, 21) were screened by fluorescence in situ hybridization (FISH). No aneuploidy was observed in 72.7% of embryos, 91% of those developed to blastocysts. Embryos were transferred on days 5-6. Clinical pregnancy was obtained in 32.8% of cases, and 60 babies were born. Patients who underwent ICSI/PGS treatment were compared with those who underwent standard ICSI treatment by examining the percentage of blastocysts, pregnancy rate, gestational length, birth height and weight. No significant differences in these parameters were observed between the groups. Day 4 biopsy procedure does not adversely affect embryo development in vitro or in vivo. The increased number of cells obtained by biopsy of morulae might facilitate diagnostic screening. There is enough time after biopsy to obtain PGD results for embryo transfer on day 5-6 in the current IVF cycle

Embryological and clinical data.

<p>Embryological and clinical data.</p

Cells obtained by biopsy for PGD.

<p>(A) Fixed nuclei stained with DAPI. (B) FISH signals for chromosome 21 (orange), the Y-chromosome (aqua), and the X-chromosome (green).</p

PGS cycle data.

<p>PGS cycle data.</p

ICSI/PGS and ICSI children born.

<p>ICSI/PGS and ICSI children born.</p

Fusion of three blastomeres inside 4-cell embryos.

<p>(A) 4-cell embryos with visible contacts between three blastomeres. (B) Embryo with one normal and one big blastomere from three fused blastomeres. (C) Hoechst 33342 staining confirms the presence of three nuclei in one cytoplast. (D) In vitro development of 4-cell embryos with three fused blastomeres. Middle stage and expanded blastocysts.</p

Oocyte-oocyte fusion and oocyte-blastomere fusion.

<p>(A) Two aggregated oocytes before fusion. (B) Fused oocytes, 60 min after laser treatment. (C) Development of fused oocytes after parthenogenetic activation and in vitro cultivation. (D) Laser irradiation of contact site between aggregated oocyte and blastomere (arrow show point of laser beams area of fusion). (E) Fused cells, 60 min after laser treatment. (F) Asymmetric cleavage of fused cells.</p

Fusion of two blastomeres inside 4-cell embryos.

<p>(A) 3-cell embryos as a result of fusion of two blastomeres inside 4-cell embryos. (B) Hoechst 33342 staining demonstrates two nuclei in one cytoplast. (C) Hatching blastocysts.</p

Laser Fusion of Mouse Embryonic Cells and Intra-Embryonic Fusion of Blastomeres without Affecting the Embryo Integrity

Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo’s integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development