Michal Kubelka

Butyrolactone I Reversibly Inhibits Meiotic Maturation of Bovine Oocytes,Without Influencing Chromosome Condensation Activity

Abstract In this study, butyrolactone I (BL I), a potent and specific inhibitor of cyclin-dependent kinases, was shown to block germinal vesicle (GV) breakdown (GVBD) in bovine oocytes in a concentration-dependent manner; GVBD was almost totally inhibited over the course of 24–48 h of culture when 100 μM BL I was included in tissue culture medium 199 containing either polyvinyl alcohol or BSA. Correlated with this inhibition was the failure of either p34cdc2 kinase or mitogen-activated protein (MAP) kinase to become activated, and it was unlikely that BL I directly inhibited MAP kinase, since 100 μM BL I did not inhibit MAP kinase activity present in extracts obtained from metaphase II-arrested bovine eggs that possess high levels of MAP kinase activity. Nevertheless, the formation of highly condensed bivalents was observed in 78% of the BL I-treated GV-intact oocytes. This result suggests that chromosome condensation during first meiosis in bovine oocytes does not require the activity of either p34cdc2 kinase or MAP kinase. Treatment of BL I-arrested oocytes with okadaic acid (OA) did not result in either the activation of p34cdc2 kinase or MAP kinase, or inducement of GVBD. The BL I-induced block of GVBD for 24 h was reversible, and a subsequent 24-h culture resulted in 90% of oocytes reaching metaphase II with emission of the first polar body. Correlated with the progression to and arrest at metaphase II was the full activation of both p34cdc2 and MAP kinases. The reversibility after 48 h of culture in BL I was partially decreased when compared to that achieved after an initial 24-h culture. Fertilization in vitro of these eggs resulted in a high incidence of both sperm penetration and pronucleus formation (88% and 70%, respectively).

PKB/AKT is involved in resumption of meiosis in mouse oocytes

Background information. In fully grown mouse oocytes, a decrease in cAMP concentration precedes and is linked to CDK1 (cyclin‐dependent kinase 1) activation. The molecular mechanism for this coupling, however, is not defined. PKB (protein kinase B, also called AKT) is implicated in CDK1 activation in lower species. During resumption of meiosis in starfish oocytes, MYT1, a negative regulator of CDK1, is phosphorylated by PKB in an inhibitory manner. It can imply that PKB is also involved in CDK1 activation in mammalian oocytes.

Protein Patterns of Pig Oocytes During In Vitro Maturation

Abstract In vitro maturation (IVM) of fully grown mammalian oocytes is characterized by initial germinal vesicle (GV) breakdown and rearrangement of microtubule network during the first meiosis (MI), followed by extrusion of the first polar body and block of the oocytes in metaphase of the second meiosis (MII). Only fully matured oocytes are capable of undergoing fertilization and the initiation of zygotic development. These observations are mostly based on morphological evaluation; however, the molecular events responsible for these processes are not known. In this study, we have launched the analysis of pig oocytes during in vitro maturation using a proteomics approach. First, oocyte proteins have been separated by two-dimensional gel electrophoresis and identified by mass spectrometry. Remarkably, several proteins, including peroxiredoxins, ubiquitin carboxyl-terminal hydrolase isozyme L1, and spermine synthase, are even more abundant than actin, usually the most abundant protein in somatic cells. Furthermore, we have initiated comparative analysis of the oocytes at different stages of maturation to characterize candidate proteins, which are differentially expressed during in vitro maturation. To date, we have identified antiquitin (D7A1), the member of aldehyde dehydrogenase family7 that has been significantly increased in MI and MII stages compared with GV oocytes. To our knowledge, this is the first pig oocyte proteome available so far that may be used as a reference map. The proteins that are differentially regulated during IVM may present potential biomarkers of oocyte maturation and quality. It is a useful inventory toward a deeper understanding of the mechanisms underlying reproduction and development.

Aurora kinase A controls meiosis I progression in mouse oocytes

Aurora kinase A (AURKA), which is a centrosome-localized serine/threonine kinase crucial for cell cycle control, is critically involved in centrosome maturation and spindle assembly in somatic cells. Active T288 phosphorylated AURKA localizes to the centrosome in the late G2 and also spreads to the minus ends of mitotic spindle microtubules. AURKA activates centrosomal CDC25B and recruits cyclin B1 to centrosomes. We report here functions for AURKA in meiotic maturation of mouse oocytes, which is a model system to study the G2 to M transition. Whereas AURKA is present throughout the entire GV-stage oocyte with a clear accumulation on microtubule organizing centers (MTOC), active AURKA becomes entirely localized to MTOCs shortly before germinal vesicle breakdown. In contrast to somatic cells in which active AURKA is present at the centrosomes and minus ends of microtubules, active AURKA is mainly located on MTOCs at metaphase I (MI) in oocytes. Inhibitor studies using Roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor) reveal that activation of AURKA localized on MTOCs is independent on PI3K-PKB and CDK1 signaling pathways and MOTC amplification is observed in roscovitine- and SH-6- treated oocytes that fail to undergo nuclear envelope breakdown. Moreover, microinjection of Aurka mRNA into GV-stage oocytes cultured in 3-isobutyl-1-methyl xanthine (IBMX)-containing medium to prevent maturation also results in MOTC amplification in the absence of CDK1 activation. Over-expression of AURKA also leads to formation of an abnormal MI spindle, whereas RNAi-mediated reduction of AURKA interferes with resumption of meiosis and spindle assembly. Results of these experiments indicate that AURKA is a critical MTOC-associated component involved in resumption of meiosis, MTOC multiplication, proper spindle formation and the metaphase I-metaphase II transition.

CDC25A phosphatase controls meiosis I progression in mouse oocytes

CDK1 is a pivotal regulator of resumption of meiosis and meiotic maturation of oocytes. CDC25A/B/C are dual-specificity phosphatases and activate cyclin-dependent kinases (CDKs). Although CDC25C is not essential for either mitotic or meiotic cell cycle regulation, CDC25B is essential for CDK1 activation during resumption of meiosis. Cdc25a -/- mice are embryonic lethal and therefore a role for CDC25A in meiosis is unknown. We report that activation of CDK1 results in a maturation-associated decrease in the amount of CDC25A protein, but not Cdc25a mRNA, such that little CDC25A is present by metaphase I. In addition, expression of exogenous CDC25A overcomes cAMP-mediated maintenance of meiotic arrest. Microinjection of Gfp-Cdc25a and Gpf-Cdc25b mRNAs constructs reveals that CDC25A is exclusively localized to the nucleus prior to nuclear envelope breakdown (NEBD). In contrast, CDC25B localizes to cytoplasm in GV-intact oocytes and translocates to the nucleus shortly before NEBD. Over-expressing GFP-CDC25A, which compensates for the normal maturation-associated decrease in CDC25A, blocks meiotic maturation at MI. This MI block is characterized by defects in chromosome congression and spindle formation and a transient reduction in both CDK1 and MAPK activities. Lastly, RNAi-mediated reduction of CDC25A results in fewer oocytes resuming meiosis and reaching MII. These data demonstrate that CDC25A behaves differently during female meiosis than during mitosis, and moreover, that CDC25A has a function in resumption of meiosis, MI spindle formation and the MI-MII transition. Thus, both CDC25A and CDC25B are critical for meiotic maturation of oocytes.

Lack of response to unaligned chromosomes in mammalian female gametes

Chromosome segregation errors are highly frequent in mammalian female meiosis, and their incidence gradually increases with maternal age. The fate of aneuploid eggs is obviously dependent on the stringency of mechanisms for detecting unattached or repairing incorrectly attached kinetochores. In case of their failure, the newly formed embryo will inherit the impaired set of chromosomes, which will have severe consequences for its further development. Whether spindle assembly checkpoint (SAC) in oocytes is capable of arresting cell cycle progression in response to unaligned kinetochores was discussed for a long time. It is known that abolishing SAC increases frequency of chromosome segregation errors and causes precocious entry into anaphase; SAC, therefore, seems to be essential for normal chromosome segregation in meiosis I. However, it was also reported that for anaphase-promoting complex (APC) activation, which is a prerequisite for entering anaphase; alignment of only a critical mass of kinetochores on equatorial plane is sufficient. This indicates that the function of SAC and of cooperating chromosome attachment correction mechanisms in oocytes is different from somatic cells. To analyze this phenomenon, we used live cell confocal microscopy to monitor chromosome movements, spindle formation, APC activation and polar body extrusion (PBE) simultaneously in individual oocytes at various time points during first meiotic division. Our results, using oocytes from aged animals and interspecific crosses, demonstrate that multiple unaligned kinetochores and severe congression defects are tolerated at the metaphase to anaphase transition, although such cells retain sensitivity to nocodazole. This indicates that checkpoint mechanisms, operating in oocytes at this point, are essential for accurate timing of APC activation in meiosis I, but they are insufficient in detection or correction of unaligned chromosomes, preparing thus conditions for propagation of the aneuploidy to the embryo.

Antibody Microarray Analyses of Signal Transduction Protein Expression and Phosphorylation during Porcine Oocyte Maturation

Kinex antibody microarray analyses was used to investigate the regulation of 188 protein kinases, 24 protein phosphatases, and 170 other regulatory proteins during meiotic maturation of immature germinal vesicle (GV+) pig oocytes to maturing oocytes that had completed meiosis I (MI), and fully mature oocytes arrested at metaphase of meiosis II (MII). Increases in apparent protein levels of protein kinases accounted for most of the detected changes during the GV to MI transition, whereas reduced protein kinase levels and increased protein phosphorylation characterized the MI to MII transition. During the MI to MII period, many of the MI-associated increased levels of the proteins and phosphosites were completely or partially reversed. The regulation of these proteins were also examined in parallel during the meiotic maturation of bovine, frog, and sea star oocytes with the Kinex antibody microarray. Western blotting analyses confirmed altered expression levels of Bub1A, IRAK4, MST2, PP4C, and Rsk2, and the phosphorylation site changes in the kinases Erk5 (T218 + Y220), FAK (S722), GSK3-beta (Y216), MEK1 (S217 + S221) and PKR1 (T451), and nucleophosmin/B23 (S4) during pig oocyte maturation.

Neither Aurora B Activity nor Histone H3 Phosphorylation Is Essential for Chromosome Condensation During Meiotic Maturation of Porcine Oocytes

Abstract Aurora kinase B (AURKB) is a chromosomal passenger protein that is essential for a number of processes during mitosis. Its activity is regulated by association with two other passenger proteins, INCENP and Survivin, and by phosphorylation on Thr 232. In this study, we examine expression and phosphorylation on Thr-232 of AURKB during meiotic maturation of pig oocytes in correlation with histone H3 phosphorylation and chromosome condensation. We show that histone H3 phosphorylation on Ser-10, but not on Ser-28, correlates with progressive chromosome condensation during oocyte maturation; Ser-10 phosphorylation starts around the time of the breakdown of the nuclear envelope, with the maximal activity in metaphase I, whereas Ser-28 phosphorylation does not significantly change in maturing oocytes. Treatment of oocytes with 50 μM butyrolactone I (BL-I), an inhibitor of cyclin-dependent kinases, or cycloheximide (10 μg/ml), inhibitor of proteosynthesis, results in a block of oocytes in the germinal vesicle stage, when nuclear membrane remains intact; however, condensed chromosome fibers or highly condensed chromosome bivalents can be seen in the nucleoplasm of BL-I- or cycloheximide-treated oocytes, respectively. In these treated oocytes, no or only very weak AURKB activity and phosphorylation of histone H3 on Ser-10 can be detected after 27 h of treatment, whereas phosphorylation on Ser-28 is not influenced. These results suggest that AURKB activity and Ser-10 phosphorylation of histone H3 are not required for chromosome condensation in pig oocytes, but might be required for further processing of chromosomes during meiosis.

Activation of bovine oocytes by specific inhibition of cyclin-dependent kinases.

Activation of bovine oocytes by experimental procedures that closely mimic normal fertilization and allow to obtain haploid oocytes is essential both for intracytoplasmic sperm injection (ICSI) and for nuclear transfer. Therefore, with the goal of producing haploid activated oocytes, this study evaluated whether bohemine, either alone or in combination with ionomycin, is able to activate young matured bovine oocytes. Furthermore, the effect of bohemine on the patterns of DNA synthesis after pronuclear formation as well as changes in histone H1 kinase and MAP kinase activities during the process of activation were studied. Our results with bohemine show that the specific inhibition of CDKs in metaphase II bovine oocytes induces parthenogenetic activation in a dose‐dependent manner (25, 50, and 100 μM, respectively), either alone (3%, 30%, and 50%) or in combination with ionomycin (30%, 70%, and 87.5%). A single pronucleus and extrusion of the second polar body was observed (97%) when Ca2+ influx was stimulated in the presence of bohemine, although pronuclear formation without polar body extrusion was observed when bohemine was used alone. Bohemine‐activated oocytes started to synthesize DNA in the first hour (37%) after their removal from bohemine‐supplemented medium (6–7 hr post‐activation; hpa). A high synchrony in the S‐phase was registered with more than 85% of parthenotes actively synthesizing DNA 8 hpa. By contrast, DNA synthesis was absent in oocytes cultured for 4, 6, and 8 hpa in the presence of bohemine and a low rate was observed by those cultured for 18 hr (30%) in bohemine‐supplemented medium. This confirms the ability of the inhibitor to arrest the cell cycle in the G1/S boundary for at least 8 hr. A drop in histone H1 kinase activity was observed in bohemine‐activated oocytes. The activity of MBP kinase decreased later than histone H1 kinase and even 4 hr after inomycin‐bohemine treatment at least half of this activity was still detectable. Then, the MBP kinase activity decreased and the lowest level could be seen 6–8 hpa. In summary, our study shows that in vitro matured bovine oocytes can be successfully activated by a synthetic inhibitor of CDKs. This effect can be improved by combination with ionomycin. The targeting of CDKs in the way to activate bovine oocytes can be an approach to improve the efficiency of mammalian oocyte activation. Mol. Reprod. Dev. 55:422–432, 2000.

Akt (protein kinase B) is implicated in meiotic maturation of porcine oocytes.

The aim of this study was to investigate the involvement of the serine/threonine protein kinase AKT (also called protein kinase B) in the control of meiosis of porcine denuded oocytes (DOs) matured in vitro. Western blot analysis revealed that the two principal AKT phosphorylation sites, Ser473 and Thr308, are phosphorylated at different stages of meiosis. In freshly isolated germinal vesicle (GV)-stage DOs, Ser473 was already phosphorylated. After the onset of oocyte maturation, the intensity of the Ser473 phosphorylation increased, however, which declined sharply when DOs underwent GV breakdown (GVBD) and remained at low levels in metaphase I- and II-stage (MI- and MII-stage). In contrast, phosphorylation of Thr308 was increased by the time of GVBD and reached maximum at MI-stage. A peak of AKT activity was noticed around GVBD and activity of AKT declined at MI-stage. To assess the role of AKT during meiosis, porcine DOs were cultured in 50 microM SH-6, a specific inhibitor of AKT. In SH-6-treated DOs, GVBD was not inhibited; on the contrary, a significant acceleration of meiosis resumption was observed. The dynamics of the Ser473 phosphorylation was not affected; however, phosphorylation of Thr308 was reduced, AKT activity was diminished at the time of GVBD, and meiotic progression was arrested in early MI-stage. Moreover, the activity of the cyclin-dependent kinase 1 (CDK1) and MAP kinase declined when SH-6-treated DOs underwent GVBD, indicating that AKT activity is involved in the regulation of CDK1 and MAP kinase. These results suggest that activity of AKT is not essential for induction of GVBD in porcine oocytes but plays a substantial role during progression of meiosis to MI/MII-stage.