laboratory of developmental biology
Developmental biology lab focuses on zygotic gene activation (ZGA) and gene expression during early embryonal development of mammals. We are using as a model early mouse and bovine embryos and bovine fibroblasts, cultivated in vitro.
Preimplantation development of mammals is characterised by three major developmental transitions, which occur after fertilisation of oocyte – transition from oogenetic to embryonic genomic control (or zygotic gene activation – ZGA), compaction (which results in the formation of polarised epithelium) and differentiation of the morula into the blastocyst The ZGA exhibits a pattern that includes the degradation of maternal mRNA in co-ordination with the onset of transcription and subsequent translation of mRNA from the embryonic genome. This switch occurs very early at mice; zygotic genome is fully activated at the stage of 2 cells. This major activation is even preceded by transcription starting already in male pronucleus at one cell stage. Similar situation was found even in bovine embryo where genome is fully activated by the late 8–cell stage and there is some evidence that minor activation is already turned on at the 2–cell stage. In contradistinction to a mouse, where reprogramming of gene expression during a preimplantation development has been well described, there is still remaining a variety of questions at cattle. Solution of this questions represents an aim of the grant GA CR 204/02/1145 “Regulation of transcriptional activity during preimplantation development of mammals.” In previous experiments we found genes (molecular markers), which express in the early embryonic development of bovines. The aim of this project is to find out, how is synthesis of these mRNAs controlled during the embryonic development of bovines (through inhibitors of the cell cycle and inhibitors of protein kinase). We shall find out by comparison of embryonic development of mouse and bovines, whether expression of some genes is evolutionarily conserved. GA CR 524/02/1135 “Correlation of gene expression and development of early bovine embryos. Comparison of embryos developing from the defined oocytes.” We propose to apply restriction fragment differential display method RT PCR (RF DD-RT PCR) on the field of early embryonic bovine development. This methodology uses the fluorescently labeled primers so it is possible to eliminate majority of the work with the radioactivity. The genes (molecular markers), found by this method, shall be used for the quality testing of bovine embryos derived from oocytes isolated from different categories of defined folicles. This enables us to study the relationship between the gene expression and developmental potential of oocytes after in vitro fertilization.
The result of these projects will be general knowledge about transcription control during the early embryonic development of mammals. It will be possible to use characterised molecular markers for testing, whether the embryo development in conditions in vitro proceeds in a normal way.
In our laboratory, we have studied an effect of epidermal growth factor (EGF) on porcine cumulus cells in different stages of foliculogenesis. The results showed that EGF stimulates proliferation of cumulus cells isolated from small and medium follicles whereas it stimulates remodeling of cytoskeleton and synthesis of hyaluronic acid in cumulus cells from large pre-ovulatory follicles. We have proved that the differences in EGF effect on cumulus cells are associated with different level of phosphorylation of EGF-receptors after ligand binding.
Next, we have studied expression of growth differentiation factor-9 (GDF-9) in porcine ovarian tissue. In rodents, GDF-9 is expressed specifically in oocytes throughout oogenesis. In pre-antral follicles, GDF-9 regulates proliferation of granulosa cells. In antral follicles, it regulates expression of different proteins, like LH-receptors and enzymes involved in production of hyaluronic acid and prostaglandines. Our results indicate that , in contrast to rodents, GDF-9 is not expressed exclusively in oocytes in the ovary, but also in somatic follicular cells. We suppose that these differences reflect different anatomical proportions of ovarian follicles in rodents and large animal species. In small follicles of rodents (200µm in diameter), the residing oocyte can control overall environment of the follicle. However, this is not possible in large porcine preovulatory follicles (10 000 µm in diameter). Therefore, somatic cells in the follicles contribute to regulation of expansion and ovulation by secretion of paracrine factors.
Finally, we have been interested in culture of pig embryos with the aim to manage the hole pre-implantation development from zygote to blastocyst stage under in vitro conditions. This is a prerequisite for introduction of new biotechnology and biomedicine methods into laboratory practice. Based on the literature studies and our own experience, we have chosen the medium NCSU 23 that enables development of pig embryos through the critical 4-cell stage in which activation of embryonic genome occurs. Pig embryos were isolated by flushing of oviducts and cultured in NCSU medium for 5-6 days. Embryos isolated in one- and 4-cell stage developed to blastocysts in 81 and 87 %, respectively. These results confirmed suitability of the medium for culture of pig pre-implantation embryos in vitro.
Address:
UZFG AV CR, v.v.i.
Laborator vyvojove biologie
Rumburska 89
277 21 Libechov
Czech Republic
staff of laboratory
)
)