The scientific activities of the Laboratory of Biochemistry and Molecular Biology of Germ Cells are focused mainly on the area of mammalian oocyte maturation and activation, as well as on the biochemical characterization of these processes. Mammalian oocytes are also utilised as a model for the study of mechanisms involved in the regulation of the cell cycle.
Meiotic maturation of mammalian oocytes is characterized by the initial block in the dictyate stage of first meiotic prophase, which is released either by a hormonal stimulus in vivo, or spontaneously, after releasing the oocytes from the follicle and subsequent culture in vitro. In the beginning of maturation the oocytes remain in the so called „germinal vesicle“ (GV) stage with an intact nuclear membrane, during maturation the nuclear membrane breaks down („germinal vesicle breakdown“, GVBD), the oocytes proceed through the first meiotic division and after extrusion of the first polar body they are blocked again in the metaphase of the second meiotic division (M II) until fertilization, or artificial activation by chemical or physical stimuli. Relatively long time of the whole process of meiotic maturation (from 12 h in the mouse to 44 h in the pig) together with high level of synchronization of oocytes in particular stages of maturation makes from mammalian oocytes a very good model for the study of processes occurring during M-phase of the cell cycle.
Our laboratory focuses mainly on the study of mechanisms involved in the regulation of initiation of meiotic maturation, and on the study of molecular basis of the main morphological changes occurring during maturation, such as breakdown of the nuclear membrane, chromosome condensation, formation of the metaphase plate and subsequent segregation of chromosomes.
In our laboratory the activation pattern of the two major M-phase kinases – cdk1 and MAP kinase during maturation of pig and cattle oocytes and after their artificial activation have been described. Our results show that both kinases become activated approximately at the time of GVBD. While the activity of cdk1 kinase (which is a subunit of the so called M-phase promoting factor, MPF) partially drops on the anaphase I/telophase I transition and then rises again to reach a maximum in metaphase II, the activity of MAP kinase remains high until metaphase II, her it is involved in the MII block as a part of the so called “cytostatic factor“, CSF. After fertilization or activation of oocytes activities of the both kinases gradually drop down. Further, our results show that neither cdk1 kinase, nor MAP kinase activities are required for the process of chromosome condensation in mammalian oocytes.

The role of another M-phase specific protein kinase, Aurora B in the process of chromosome condensation during meiotic maturation of pig oocytes has been also studied recently in our laboratory. We have proved that neither Aurora B kinase, nor phosphorylation of its substrate, histone H3 is essential for chromosome condensation; these activities are important rather in the later stages of maturation, most likely for the correct organization of the chromosomes in the meiotic spindle and for the segregation of the chromosomes during anaphase I.
At the present we concentrate on the study of the role of protein kinase B (Akt kinase, PKB) in the regulation of initiation of the meiotic maturation of mouse and pig oocytes. The first results in the mouse have shown that the activation of PKB in mouse oocytes is essential for cdk1 kinase activation, i.e. also for the resumption of meiotic maturation. During maturation a dynamic relocalisation of PKB occurs in the oocytes, which is dependent on the degree of PKB phosphorylation on T308 and S473 residues. At least two populations of PKB can be identified in the oocytes, which differ in localization and phosphorylation on T308 and S473, however, PKB phosphorylated on both sites can be found only on centrosome (MTOC) during breakdown of the nuclear membrane.
The role of the above mentioned MAP kinase (Erk1/Erk2) during mammalian oocyte maturation (apart from its importance in maintaining the M II block as a part of CSF) hasn’t been described. Our recent results show that Erk1/Erk2 MAP kinase is with high probability involved in phosphorylation and activation of Mnk1 and Mnk2 protein kinases (MAP kinase-interacting kinases 1 and 2), the kinases responsible for phosphorylation of translation initiation factor eIF4E. During meiotic maturation of pig oocytes eIF4E becomes gradually phosphorylated. Similarly, gradual phosphorylation of its regulatory protein, 4E-BP1 occurs in this period, which is at least partly mediated by mTOR/FRAP protein kinase. As a result of these phosphorylation changes, eIF4E becomes to be bound to eIF4G and an active translation initiation complex, eIF4F is formed. In such a way the cap-dependent mechanism of translation initiation is activated during meiotic maturation of pig oocytes, however, the overall translation rates gradually decrease in this period. We are currently focusing on explanation of these findings, i.e. we are testing two following hypotheses: first one would suggest that during pig oocyte IVM, the most of the translation is not dependent on the cap and that the cap-dependent mechanisms are required only for translation of specific mRNAs; second hypothesis is based on the possible presence of another mechanism inhibiting cap-dependent initiation of translation in MI and MII stage oocytes (e.g. other initiation factors, or PABP, PAIP1,2 proteins).
Proteome group of Laboratory of Biochemistry and Molecular Biology of germ cells is involved in analysis of oocyte proteomes during meiotic maturation using classical proteomics approach (2-DE and MS). Current studies demonstrate major prevalence of redox- regulating proteins (37% of all identified proteins) and extremely high level of other proteins including Ubiquitin C-terminal hydrolase isoenzym L1. Further work is focused on phosphoproteome of maturing mammalian oocytes as a fine regulatory mechanism underlying developmental processes.
Proteomics approach is used to study of proteomes and phosphoproteomes induced by inhibition of cellular cyclin-dependent kinases, a new potential generation of anticancer drugs.
Our recent findings demonstrating down regulation of three histone variants indicated that anti-mitotic and anti-cancer activities of this compounds might be associated with epigenetic regulation at the level of chromatin structure.

Recently, the Joint Laboratory of Proteome Analysis was established between our laboratory and laboratories of Institute of Microbiology AS CR, Institute of Experimental Medicine AS CR, and Immunotech a.s. This cooperation allows the expansion of our current proteome analyses from 2-D gel electrophoretic techniques to multidimensional chromatographic protein separations (ProteomeLab PF 2D, Beckman Coulter) in order to obtain comprehensive view of studied biological system.