Developmental biology laboratory focuses on gene and protein expression during oocyte meiotic maturation and early embryonic development of mammals. We are using as a model pig oocytes and bovine oocytes and embryos, cultivated in vitro.

During the growth phase, the oocyte is arrested in the first meiotic prophase, but it gains full meiotic competence in several steps. However, these meiotically competent oocytes still remain in the dictyate stage of the prophase if they are retained within follicles. They only resume meiosis following the preovulatory surge of luteinizing hormone (LH) or after the release into a suitable culture medium. The resumption of meiosis is regulated inside oocytes by a post-translation mechanism and does not depend on the activation of genes in the oocyte itself. Nevertheless, the LH-induced meiotic resumption is accompanied by a dramatic change in gene expression profiles in mural granulosa and cumulus cells. The changes in the cumulus/granulosa cell transcriptome are under the control of a broad signaling network activated in the follicular cells by the LH surge.

Subsequent preimplantation development of mammals is characterized by three major developmental transitions, which occur after fertilization of oocyte – transition from oogenetic to embryonic genomic control (or zygotic gene activation – ZGA), compaction (which results in the formation of polarized 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 oocyte meiotic maturation and reprogramming of gene expression has been well described, there is still remaining a variety of questions at porcine and bovine species, which represent better model for human oogenesis and embryogenesis than rodents.

We currently concentrate on two main areas, which are studied in our laboratory:

Pig oocyte meiotic maturation

We have been studying molecular mechanisms regulating oocyte meiotic arrest and meiotic resumption with a special attention to somatic follicular cell and oocyte intercellular communication. By the use of real-time polymerase chain reaction, microarray analysis and immunoblotting, we have identified on a model of pig cumulus-oocyte complexes genes and proteins that are involved in regulation of oocyte meiotic maturation. A special attention was paid to characterization of signaling molecules and signaling cascades that are activated in preovulatory follicles by gonadotropin hormones and modified by locally produced growth factors. 

 Role of cyclic guanosine monophosphate (cGMP)

 Epidermal growth factor (EGF)-like peptide

(1) Pig oocyte-cumulus complexes cultured in vitro in medium with dbcAMP.

(2) Expanded oocyte-cumulus complex isolated from pig pre-ovulatory follicle 24 h after application of hCG.

(1) Assessment of gene expression by microarrays.

(2) Assessment of mitogen-activated protein kinase activity in pig oocyte-cumulus complexes cultured in medium with FSH and 8-CPT-cGMP.

Bovine preimplantation development

Role of nucleophosmin

Nucleophosmin was for our study chosen as a gene potentially crucially important for bovine preimplantation development. We have found that embryonic transcription of nucleophosmin starts during the major wave of embryonic genome activation which indicates its necessity for bovine preimplantation development. The mRNA expression level of nucleophosmin was found to decrease from MII stage to early 8-cell stage and started to increase at late 8-cell stage. This increase was α-amanitin sensitive and thus represents the start of transcription from embryonic genome. Consequently, we monitored the influence of nucleophosmin mRNA silencing on the early embryogenesis. In summary our data have shown that a small amount of nucleophosmin protein is preserved throughout whole preimplantation development and enables the development of embryos until the blastocyst stage.

SCF complex

Currently, our most important research issue concerns ubiquitination during bovine preimplantation development. We concentrate on Skp1-Cullin1-Fbox (SCF) complex, the expression of its invariant components (Cullin 1, Skp1, Rbx1) and its contribution to degradation of maternal proteins. Our up to now results have shown that embryonic expression of all these genes starts in initial stages of development. Especially Cullin 1 is activated very early, already at 4-cell stage. Genes participating in ubiquitination are usually activated at 8-cell stage, and the early activation of Cullin 1 suggests its necessity for embryonic genome activation. Protein localization analysis showed interesting results especially at the blastocyst stage. There was clear concentration of protein expression and SCF complex activation to trophectoderm. These results allowed us to initiate the experiments dealing with the application of SCF complex during preimplantation development and especially in maternal protein degradation.

Confocal laser scanning microscopy of bovine blastocyst after Duolink In situ PLA analysis. PLA signal indicates CUL1 – SKP1 interactions (red), nuclei (blue).

Cover: Confocal laser scanning microscopy of tubulin polymerization of bovine embryo after immunofluorescence analysis. Alpha-tubulin (green), nucleophosmin (red), nuclei (blue). Toralová et al. 2012, Bovine preimplantation embryos with silenced nucleophosmin mRNA are able to develop until the blastocyst stage. Reproduction 144 349-359.

Adress:

IAPG AS CR, v.v.i.
Laboratory of Developmental Biology
Rumburská 89
277 21 Liběchov
Czech Republic