Involvement of calcium/calmodulin-dependent protein kinase kinase in meiotic maturation of pig oocytes

Calcium (Ca(2+))/calmodulin-dependent protein kinase kinase (CaMKK) is a novel member of Ca(2+)/calmodulin-dependent protein kinase (CaMK) family, whose physiological roles in regulating meiotic cell cycle needs to be determined. We showed by Western blot that CaMKK was expressed in pig oocytes at various maturation stages. Confocal microscopy was employed to observe CaMKK distribution. In oocytes at the germinal vesicle (GV) or prometaphase I (pro-MI) stage, CaMKK was distributed in the nucleus, around the condensed chromatin and the cortex of the cell. At metaphase I (MI) stage, CaMKK was concentrated in the cortex of the cell. After transition to anaphase I or telophase I stage, CaMKK was detected around the separating chromosomes and in the cortex of the cell. At metaphase II (MII) stage, CaMKK was localized to the cortex of the cell, with a thicker area near the first polar body (PB1). Treatment of pig cumulus-enclosed oocytes with STO-609, a membrane-permeable CaMKK inhibitor, resulted in the delay/inhibition of the meiotic resumption and the inhibition of first polar body emission. The correlation between CaMKK and microfilaments during meiotic maturation of pig oocytes was then studied. CaMKK and microfilaments were colocalized from MI to MII during porcine oocyte maturation. After oocytes were treated with STO-609, microfilaments were depolymerized, while in oocytes exposed to cytochalasin B (CB), a microfilament polymerization inhibitor, CaMKK became diffused evenly throughout the cell. These data suggest that CaMKK is involved in regulating the meiotic cell cycle probably by interacting with microfilaments in pig oocytes.     

Arrest of cell cycle progression during first interphase in murine zygotes microinjected with anti-PCM-1 antibodies

To investigate the function of the centrosome protein PCM-1, antibodies against PCM-1 were microinjected into either germinal vesicle stage meiotic oocytes or fertilized mouse eggs, and cell cycle progression events (i.e., microtubule assembly, chromosome and centrosome organization, meiotic maturation) were assayed. These studies determined that microinjected PCM-1 antibodies arrested cell cycle progression, with anti-PCM-1 arresting fertilized eggs at the pronucleate stage when injected during G1. Analysis of the injected eggs determined that centrosome disruption and microtubule cytaster disorganization accompanied the cell cycle arrest. Anti-PCM-1 blocked neither pronuclear centration, completion of mitosis when microinjected into zygotes at G2, nor meiotic maturation when microinjected into immature oocytes. These results identify a novel role for PCM- 1 in cell cycle regulation, and indicate that PCM-1 must fulfill an essential function for cells to complete interphase.     

Aven is dynamically regulated during Xenopus oocyte maturation and is required for oocyte survival

We have analyzed the expression and function of the cell death and cell cycle regulator Aven in Xenopus. Analysis of Xenopus Aven expression in oocytes and embryos revealed a band close to the predicted molecular weight of the protein (36 kDa) in addition to two bands of higher molecular weight (46 and 49 kDa), one of which was determined to be due to phosphorylation of the protein. The protein is primarily detected in the cytoplasm of oocytes and is tightly regulated during meiotic and mitotic cell cycles. Progesterone stimulation of oocytes resulted in a rapid loss of Aven expression with the protein levels recovering before germinal vesicle breakdown (GVBD). This loss of Aven is required for the G2–M1 cell cycle transition. Aven morpholino knockdown experiments revealed that early depletion of the protein increases progesterone sensitivity and facilitates GVBD, but prolonged depletion of Aven results in caspase-3 activation and oocyte death by apoptosis. Phosphorylated Aven (46 kDa) was found to bind Bcl-x_L in oocytes, but this interaction was lost in apoptotic oocytes. Thus, Aven alters progesterone sensitivity in oocytes and is critical for oocyte survival.     

Xkid chromokinesin is required for the meiosis I to meiosis II transition in Xenopus laevis oocytes

Xkid chromokinesin is required for chromosome alignment on the metaphase plate of spindles formed in Xenopus laevis egg extracts. We have investigated the role of Xkid in Xenopus oocyte meiotic maturation, a progesterone-triggered process that reinitiates the meiotic cell cycle in oocytes arrested at the G2/M border of meiosis I. Here we show that Xkid starts to accumulate at the time of germinal vesicle breakdown and reaches its largest quantities at metaphase II in oocytes treated with progesterone. Both germinal vesicle breakdown and spindle assembly at meiosis I can occur normally in the absence of Xkid. But Xkid-depleted oocytes cannot reactivate Cdc2/cyclin B after meiosis I and, instead of proceeding to meiosis II, they enter an interphase-like state and undergo DNA replication. Expression of a Xkid mutant that lacks the DNA-binding domain allows Xkid-depleted oocytes to complete meiotic maturation. Our results show that Xkid has a role in the meiotic cell cycle that is independent from its role in metaphase chromosome alignment.     

AMPK regulation of mouse oocyte meiotic resumption in vitro

We have previously shown that the adenosine analog 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), an activator of AMP-activated protein kinase (AMPK), stimulates an increase in AMPK activity and induces meiotic resumption in mouse oocytes [Downs, S.M., Hudson, E.R., Hardie, D.G., 2002. A potential role for AMP-activated protein kinase in meiotic induction in mouse oocytes. Dev. Biol, 245, 200-212]. The present study was carried out to better define a causative role for AMPK in oocyte meiotic maturation. When microinjected with a constitutively active AMPK, about 20% of mouse oocytes maintained in meiotic arrest with dibutyryl cAMP (dbcAMP) were stimulated to undergo germinal vesicle breakdown (GVB), while there was no effect of catalytically dead kinase. Western blot analysis revealed that germinal vesicle (GV)-stage oocytes cultured in dbcAMP-containing medium plus AICAR possessed elevated levels of active AMPK, and this was confirmed by AMPK assays using a peptide substrate of AMPK to directly measure AMPK activity. AICAR-induced meiotic resumption and AMPK activation were blocked by compound C or adenine 9-beta-d-arabinofuranoside (araA, a precursor of araATP), both inhibitors of AMPK. Compound C failed to suppress adenosine uptake and phosphorylation, indicating that it did not block AICAR action by preventing its metabolism to the AMP analog, ZMP. 2'-deoxycoformycin (DCF), a potent adenosine deaminase inhibitor, reversed the inhibitory effect of adenosine on oocyte maturation by modulating intracellular AMP levels and activating AMPK. Rosiglitazone, an anti-diabetic agent, stimulated AMPK activation in oocytes and triggered meiotic resumption. In spontaneously maturing oocytes, GVB was preceded by AMPK activation and blocked by compound C. Collectively, these results support the proposition that active AMPK within mouse oocytes provides a potent meiosis-inducing signal in vitro.     

Functional specialization of chordate CDK1 paralogs during oogenic meiosis

Cyclin-dependent kinases (CDKs) are central regulators of eukaryotic cell cycle progression. In contrast to interphase CDKs, the mitotic phase CDK1 is the only CDK capable of driving the entire cell cycle and it can do so from yeast to mammals. Interestingly, plants and the marine chordate, Oikopleura dioica, possess paralogs of the highly conserved CDK1 regulator. However, whereas in plants the 2 CDK1 paralogs replace interphase CDK functions, O. dioica has a full complement of interphase CDKs in addition to its 5 odCDK1 paralogs. Here we show specific sub-functionalization of odCDK1 paralogs during oogenesis. Differential spatiotemporal dynamics of the odCDK1a, d and e paralogs and the meiotic polo-like kinase 1 (Plk1) and aurora kinase determine the subset of meiotic nuclei in prophase I arrest that will seed growing oocytes and complete meiosis. Whereas we find odCDK1e to be non-essential, knockdown of the odCDK1a paralog resulted in the spawning of non-viable oocytes of reduced size. Knockdown of odCDK1d also resulted in the spawning of non-viable oocytes. In this case, the oocytes were of normal size, but were unable to extrude polar bodies upon exposure to sperm, because they were unable to resume meiosis from prophase I arrest, a classical function of the sole CDK1 during meiosis in other organisms. Thus, we reveal specific sub-functionalization of CDK1 paralogs, during the meiotic oogenic program.     

Checkpoint kinase 1 is essential for meiotic cell cycle regulation in mouse oocytes

Checkpoint kinase 1 (Chk1) plays key roles in all currently defined cell cycle checkpoints, but its functions in mouse oocyte meiosis remain unclear. In this study, we report the expression, localization and functions of Chk1 in mouse oocyte meiosis. Chk1 was expressed from germinal vesicle (GV) to metaphase II (MII) stages and localized to the spindle from pro-metaphase I (pro-MI) to MII stages in mouse oocytes. Chk1 depletion facilitated the G₂/M transition while Chk1 overexpression inhibited the G₂/M transition as indicated by germinal vesicle breakdown (GVBD), through regulation of Cdh1 and Cyclin B1. Chk1 depletion did not affect meiotic cell cycle progression after GVBD, but its overexpression after GVBD activated the spindle assembly checkpoint and prevented homologous chromosome segregation, thus arresting oocytes at pro-MI or metaphase I (MI) stages. These results suggest that Chk1 is indispensable for prophase I arrest and functions in G₂/M checkpoint regulation in meiotic oocytes. Moreover, Chk1 overexpression affects meiotic spindle assembly checkpoint regulation and thus chromosome segregation.     

Cyclin B synthesis is required for sea urchin oocyte maturation

Sea urchins are members of a limited group of animals in which meiotic maturation of oocytes is completed prior to fertilization. This is different from oocytes of most animals such as mammals and amphibians in which fertilization reactivates an arrested meiotic cycle. Using a recently developed technique for in vitro maturation of sea urchin oocytes, we analyzed the role of cyclin B, the regulatory component of maturation-promoting factor, in the control of sea urchin oocyte meiotic induction and progression. Oocytes of the sea urchin Lytechinus variegatus accumulate significant amounts of cyclin B mRNA and protein during oogenesis. We analyzed cyclin B synthetic requirements in oocytes and early embryos by inhibiting cyclin B synthesis with DNA and morpholino antisense oligonucleotides. Cyclin B synthesis is not necessary for the entry of G2-arrested oocytes into meiosis; however, it is required for the proper progression through meiotic divisions. Surprisingly, mature sea urchin eggs contain significant cyclin B protein following meiosis that serves as a maternal store for early cleavage divisions. We also find that cyclin A can functionally substitute for cyclin B in early embryos but not in oocytes. These studies provide a foundation for understanding the mechanism of meiotic maturation independent of the zygotic cell cycle.     

Calmodulin triggers the resumption of meiosis in amphibian oocytes

The calcium-binding protein, calmodulin, has been purified from Xenopus laevis oocytes. This 18,500-dalton protein, pl 4.3, has two high-affinity calcium-binding sites per mole protein having a dissociation constant of 2.8 x 10(-6) M. Full-grown Xenopus oocytes, arrested in late G2 of the meiotic cell cycle, resumed meiosis when microinjected with 60-80 ng (3-4 pmol) of calmodulin in the form of a calcium-calmodulin complex. The timing of the meiotic events in these recipient oocytes was the same as that normally induced by progesterone. Xenopus ovarian calmodulin stimulated bovine brain phosphodiesterase (PDE) 3- to 10-fold in a calcium-dependent manner, but it had no apparent effect on ovarian PDE activity. A calcium-calmodulin-dependent protein kinase has been isolated from Xenopus oocytes using a calmodulin-Sepharose 4B affinity column. The possible role for this kinase in regulating the G2-M transition in oocytes has been discussed.     

The C.elegans MAPK phosphatase LIP-1 is required for the G(2)/M meiotic arrest of developing oocytes

In the Caenorhabditis elegans hermaphrodite germline, spatially restricted mitogen-activated protein kinase (MAPK) signalling controls the meiotic cell cycle. First, the MAPK signal is necessary for the germ cells to progress through pachytene of meiotic prophase I. As the germ cells exit pachytene and enter diplotene/diakinesis, MAPK is inactivated and the developing oocytes arrest in diakinesis (G(2)/M arrest). During oocyte maturation, a signal from the sperm reactivates MAPK to promote M phase entry. Here, we show that the MAPK phosphatase LIP-1 dephosphorylates MAPK as germ cells exit pachytene in order to maintain MAPK in an inactive state during oocyte development. Germ cells lacking LIP-1 fail to arrest the cell cycle at the G(2)/M boundary, and they enter a mitotic cell cycle without fertilization. LIP-1 thus coordinates oocyte cell cycle progression and maturation with ovulation and fertilization.     

Functions of Fyn kinase in the completion of meiosis in mouse oocytes

Oocyte maturation invokes complex signaling pathways to achieve cytoplasmic and nuclear competencies for fertilization and development. The Src-family kinases FYN, YES and SRC are expressed in mammalian oocytes but their function during oocyte maturation remains an open question. Using chemical inhibitor, siRNA knockdown, and gene deletion strategies the function of Src-family kinases was evaluated in mouse oocytes during maturation under in vivo and in vitro conditions. Suppression of Src-family as a group with SKI606 greatly reduced meiotic cell cycle progression to metaphase-II. Knockdown of FYN kinase expression after injection of FYN siRNA resulted in an approximately 50% reduction in progression to metaphase-II similar to what was observed in oocytes isolated from FYN (−/−) mice matured in vitro. Meiotic cell cycle impairment due to a Fyn kinase deficiency was also evident during oocyte maturation in vivo since ovulated cumulus oocyte complexes collected from FYN (−/−) mice included immature metaphase-I oocytes (18%). Commonalities in meiotic spindle and chromosome alignment defects under these experimental conditions demonstrate a significant role for Fyn kinase activity in meiotic maturation.     

Mitogen-activated protein kinase activity during goat oocyte maturation and the acquisition of meiotic competence

Changes in MPF and MAPK activities during meiotic maturation of goat oocytes were investigated. Detection of MPF activity occurred concomitantly with GVBD, increased at MI, decreased during anaphase-telophase I transition, and increased thereafter in MII oocytes. The appearance of MAPK activity was delayed compared to MPF activity. MAPK activity increased after GVBD and persisted during the MI-MII transition. Whether MAPK was implicated in goat oocyte meiotic competence was also investigated by using oocytes from different follicle size categories that arrest at specific stages of the maturation process (GV, GVBD, MI, and MII). Results indicate that the ability of goat oocytes to resume meiosis is not directly related to the presence of Erk2. The ability to phosphorylate MAPK is acquired by the oocyte during follicular growth after the ability to resume meiosis. GVBD-arrested oocytes exhibited a high level of MPF activity after 27 hr of culture. However, 28% of oocytes from this group contained inactive MAPK, and 72% exhibited high MAPK activity. In addition, 29% of GVBD-arrested oocytes contained a residual interphasic network without recruitment of microtubules around the condensed chromosomes; 71% of GVBD-arrested oocytes displayed recruitment of microtubules near the condensed chromosomes and contained asters of microtubules distributed throughout the cytoplasm. These results indicate that oocytes arrested at GVBD were not exactly at the same point in the meiotic cell cycle progression, and suggest that MAPK could be implicated in the regulation of microtubule organization. The data presented here suggest that in goat oocytes, MAPK is not implicated in the early events of meiosis resumption, but rather in post-GVBD events such as spindle formation and MII arrest. © 1996 Wiley-Liss Inc.     

Changes in the activity of type 2A protein phosphatases during meiotic maturation and the first mitotic cell cycle in mouse oocytes.

We have established an assay to measure protein phosphatase activity in mouse oocytes using [32P]-radiolabeled phosphorylase a as the substrate. Removal of the radiolabel from the substrate in vitro was linear with time and could be inhibited totally by the addition of okadaic acid (inhibitor of type 1 and type 2 protein phosphatases), or partially by protein inhibitor 2 (inhibitor of type 1 protein phosphatases). We performed a detailed study of the activity of type 2A protein phosphatases in mouse oocytes undergoing meiotic maturation and after parthenogenetic activation of mature oocytes arrested in metaphase II. Significant changes in the activity of type 2A protein phosphatases were observed during the first meiotic and the first mitotic cell cycles. These alterations in type 2A protein phosphatase activity occurred in the absence of changes in the quantity of the catalytic sub-unit and can be correlated with changes in the activity of protein kinases and rearrangement of the cellular cytoskeleton. Our observations support a role for type 2A protein phosphatases in cell cycle regulation and demonstrate that, like the protein kinases, the type 2A phosphatases also undergo changes in their activity during early mammalian development.     

A potential role for AMP-activated protein kinase in meiotic induction in mouse oocytes

Cyclic adenosine monophosphate (cAMP) has been implicated as an important regulator of meiotic maturation in mammalian oocytes. A decrease in cAMP, brought about by the action of cAMP phosphodiesterase (PDE), is thought to initiate germinal vesicle breakdown (GVB) by the inactivation of cAMP-dependent protein kinase. However, the product of PDE activity, 5'-AMP, is a potent activator of an important regulatory enzyme, AMP-activated protein kinase (AMPK). The aim of this study was to evaluate a possible role for AMPK in meiotic induction, using oocytes obtained from eCG-primed, immature mice. Alpha-1 and -2 isoforms of the catalytic subunit of AMPK were detected in both oocytes and cumulus cells. When 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside (AICA riboside), an activator of AMPK, was tested on denuded oocytes (DO) and cumulus cell-enclosed oocytes (CEO) maintained in meiotic arrest by dbcAMP or hypoxanthine, GVB was dose-dependently induced. Meiotic induction by AICA riboside in dbcAMP-supplemented medium was initiated within 3 h in DO and 4 h in CEO and was accompanied by increased AMPK activity in the oocyte. AICA riboside also triggered GVB when meiotic arrest was maintained with hypoxanthine, 8-AHA-cAMP, guanosine, or milrinone, but was ineffective in olomoucine- or roscovitine-arrested oocytes, indicating that it acts upstream of maturation-promoting factor. Adenosine monophosphate dose-dependently stimulated GVB in DO when meiotic arrest was maintained with dbcAMP or hypoxanthine. This effect was not mimicked by other monophosphate or adenosine nucleotides and was not affected by inhibitors of ectophosphatases. Combined treatment with adenosine and deoxycoformycin, an adenosine deaminase inhibitor, stimulated GVB in dbcAMP-arrested CEO, suggesting AMPK activation due to AMP accumulation. It is concluded that phosphodiesterase-generated AMP may serve as a transducer of the meiotic induction process through activation of AMPK.     

Oocyte-specific expression of Gpr3 is required for the maintenance of meiotic arrest in mouse oocytes

The maintenance of meiotic prophase arrest in mouse oocytes within fully grown follicles, prior to the surge of luteinizing hormone (LH) that triggers meiotic resumption, depends on a high level of cAMP within the oocyte. cAMP is produced within the oocyte, at least in large part, by the G(s)-linked G-protein-coupled receptor, GPR3. Gpr3 is localized in the mouse oocyte but is also present throughout the follicle. To investigate whether Gpr3 in the follicle cells contributes to the maintenance of meiotic arrest, RNA interference (RNAi) was used to reduce the amount of Gpr3 RNA within follicle-enclosed oocytes. Follicle-enclosed oocytes injected with small interfering double-stranded RNA (siRNA) targeting Gpr3, but not control siRNAs, stimulated the resumption of meiosis in the majority of oocytes following a 3-day culture period. Reduction of RNA was specific for Gpr3 because an unrelated gene was not reduced by microinjection of siRNA. Meiotic resumption was stimulated in isolated oocytes injected with the same siRNA and cultured for 1 to 2 days, but at a much lower rate than in follicle-enclosed oocytes that could be cultured for longer. These results demonstrate that GPR3 specifically in the oocyte, rather than in the follicle cells, is responsible for maintenance of meiotic arrest in mouse oocytes. Furthermore, the method developed here for specifically reducing RNA in follicle-enclosed oocytes, which can be cultured for a sufficient time to reduce the level of endogenous protein, should be generally useful for targeting a wide range of other proteins that may be involved in meiotic arrest, the resumption of meiosis, fertilization, or early embryonic development.     

MPF components and meiotic competence in growing pig oocytes

Growing pig oocytes (≤90 μm in diameter) are unable to resume meiosis in vitro. The objective of our present experiments has been to identify the reasons for meiotic competence in these cells. By comparing histone H1 kinase activity in growing and fully grown oocytes we demonstrate that incompetence is associated with an inability to activate H1 kinase in growing oocytes. Immunoblotting was used to determine whether this kinase inactivity resulted from a lack of either p34^cdc2 protein or B-type cyclin. The results established that each of these cell cycle molecules are present in comparable amounts in both growing and fully grown oocytes. In the third series of studies experiments were carried out in an attempt to induce p34^cdc2 activation during growth. Treatment with okadaic acid, an inhibitor of phosphatase 1 and 2A known to stimulate and accelerate the transition into M-phase of the meiotic cycle in a number of different species, was able to induce p34^cdc2 kinase activity and facilitated the G₂- to M-phase in growing oocytes. We conclude that although growing oocytes in pigs have sufficient key cell cycle components for the G₂ to M transition, they remain incapable of converting these components to active maturation-promoting factor (MPF) until growth is virtually completed. Inhibition of phosphatase 1 or 2A induces the formation of active MPF during growth by an as yet unidentified pathway. © 1994 Wiley-Liss, Inc.     

Centriolin,a centriole-appendage protein,regulates peripheral spindle migration and asymmetric division in mouse meiotic oocytes

Unlike somatic cells mitosis, germ cell meiosis consists of 2 consecutive rounds of division that segregate homologous chromosomes and sister chromatids, respectively. The meiotic oocyte is characterized by an absence of centrioles and asymmetric division. Centriolin is a relatively novel centriolar protein that functions in mitotic cell cycle progression and cytokinesis. Here, we explored the function of centriolin in meiosis and showed that it is localized to meiotic spindles and concentrated at the spindle poles and midbody during oocyte meiotic maturation. Unexpectedly, knockdown of centriolin in oocytes with either siRNA or Morpholino micro-injection, did not affect meiotic spindle organization, cell cycle progression, or cytokinesis (as indicated by polar body emission), but led to a failure of peripheral meiotic spindle migration, large polar body emission, and 2-cell like oocytes. These data suggest that, unlike in mitotic cells, the centriolar protein centriolin does not regulate cytokinesis, but plays an important role in regulating asymmetric division of meiotic oocytes.