Role of Mitogen Activated Protein Kinase and Maturation Promoting Factor During the Achievement of Meiotic Competency in Mammalian Oocytes.

The oocyte quality remains as one of the major problems associated with poor in vitro fertilization (IVF) rate and assisted reproductive technology (ART) failure worldwide. The oocyte quality is dependent on its meiotic maturation that begins inside the follicular microenvironment and gets completed at the time of ovulation in most of the mammalian species. Follicular oocytes are arrested at diplotene stage of first meiotic prophase. The resumption of meiosis from diplotene arrest, progression through metaphase-I (M-I) and further arrest at metaphase-II (M-II) are important physiological requirements for the achievement of meiotic competency in mammalian oocytes. The achievement of meiotic competency is dependent upon cyclic stabilization/destabilization of maturation promoting factor (MPF). The mitogen-activated protein kinase3/1 (MAPK3/1) modulates stabilization/destabilization of MPF in oocyte by interacting either with signal molecules, transcription and post-transcription factors in cumulus cells or cytostatic factors (CSFs) in oocyte. MPF regulates meiotic cell cycle progression from diplotene arrest to M-II arrest and directly impacts oocyte quality. The MAPK3/1 activity is not reported during spontaneous meiotic resumption but its activity in cumulus cells is required for gonadotropin-induced oocyte meiotic resumption. Although high MAPK3/1 activity is required for the maintenance of M-II arrest in several mammalian species, its cross-talk with MPF remains to be elucidated. Further studies are required to find out the MAPK3/1 activity and its impact on MPF destabilization/stabilization during achievement of meiotic competency, an important period that decides oocyte quality and directly impacts ARTs outcome in several mammalian species including human. J. Cell. Biochem. 119: 123-129, 2018. © 2017 Wiley Periodicals, Inc.

Entry into mitosis: a solution to the decades-long enigma of MPF.

Maturation or M phase-promoting factor (MPF) is the universal inducer of M phase common to eukaryotic cells. MPF was originally defined as a transferable activity that can induce the G2/M phase transition in recipient cells. Today, however, MPF is assumed to describe an activity that exhibits its effect in donor cells, and furthermore, MPF is consistently equated with the kinase cyclin B-Cdk1. In some conditions, however, MPF, as originally defined, is undetectable even though cyclin B-Cdk1 is fully active. For over three decades, this inconsistency has remained a long-standing puzzle. The enigma is now resolved through the elucidation that MPF, defined as an activity that exhibits its effect in recipient cells, consists of at least two separate kinases, cyclin B-Cdk1 and Greatwall (Gwl). Involvement of Gwl in MPF can be explained by its contribution to the autoregulatory activation of cyclin B-Cdk1 and by its stabilization of phosphorylations on cyclin B-Cdk1 substrates, both of which are essential when MPF induces the G2/M phase transition in recipient cells. To accomplish these tasks, Gwl helps cyclin B-Cdk1 by suppressing protein phosphatase 2A (PP2A)-B55 that counteracts cyclin B-Cdk1. MPF, as originally defined, is thus not synonymous with cyclin B-Cdk1, but is instead a system consisting of both cyclin B-Cdk1 that directs mitotic entry and Gwl that suppresses the anti-cyclin B-Cdk1 phosphatase. The current view that MPF is a synonym for cyclin B-Cdk1 in donor cells is thus imprecise; instead, MPF is best regarded as the entire pathway involved in the autoregulatory activation of cyclin B-Cdk1, with specifics depending on the experimental system.

Brilliant Cresyl Blue stain selects largest oocytes with highest mitochondrial activity, maturation-promoting factor activity and embryo developmental competence in prepubertal sheep.

The aim of this study was to test the Brilliant Cresyl Blue (BCB) stain to select prepubertal sheep oocytes for in vitro blastocyst production. Oocyte diameter, mitochondrial activity, maturation-promoting factor (MPF) activity and mRNA relative expression (RE) of genes related to metabolism (ATPase Na(+)/K(+) transporting α 1 (ATP1A1) and cytochrome c oxidase subunit 1 (COX1)) and constitutive function of the cell (cytoplasmic polyadenylation-element-binding protein (CPEB) and S100A10) were assessed. Immature oocytes were exposed to different BCB concentrations (13, 26, 39 and 52  µM) and classified according to their cytoplasm colouration as grown BCB+ (blue cytoplasm) and growing BCB- (colourless cytoplasm). Staining oocytes with 13  µM BCB during 60  min allows selection of (BCB+) the largest (123.66  µm) and most competent oocytes to develop to the blastocyst stage (21%) with a higher number of cells (69.71 ± 6.19 s.e.m.) compared with non-stained BCB- oocytes (106.82  µm, 9% and 45.91 ± 3.35 s.e.m. respectively). Mitochondrial activity, assessed by MitoTracker Orange CMTMRos probe, was significantly higher in BCB+ than in BCB- oocytes after in vitro maturation (3369 and 1565  AU respectively). MPF activity was assessed by CDC2 kinase activity assay showing significantly higher activity at metaphase II stage in BCB+ than in BCB- oocytes (1.479 ± 0.09 and 1.184 ± 0.05 optical density respectively). The genes analysed in this work, ATP1A1, COX1, CPEB and S100A 10, did not show significant effect in mRNA RE between BCB selected oocytes. In conclusion, BCB stains larger and more competent oocytes to develop to the blastocyst stage with more active mitochondria and MPF activity and higher blastocyst cell number.

Cdc2 regulatory factors.

A growing family of kinases and phosphatases controls the activity of the cyclin-dependent kinase cdc2. The past year has seen the identification of the cdk activating kinase as well as considerable elucidation of the cdc25/wee1 regulatory pathways. Both cdc25 and wee1 appear to be regulated by upstream kinase/phosphatase networks. In addition, it is likely that other regulatory mechanisms cooperate with the wee1/cdc25 phosphorylation systems to control the action of cdc2. Together, these elaborate checks and balances ensure that cdc2 triggers mitosis at the appropriate time.

Regulating the onset of mitosis.

In eukaryotes, G2/M progression is mediated by activation of mitosis promoting factor (MPF). To ensure faithful chromosome segregation, the activity of key mitotic inducers and inhibitors are coupled with chromosome replication, spindle pole duplication, morphogenesis, and DNA damage. Evidence gathered in the past two years has underscored the importance of positioning MPF and its regulators in the proper place at the proper time to ensure orderly progression through the G2/M transition. Altering the spatial organization of G2/M regulators also contributes to prevention of mitosis following DNA damage.

Effects of puberty and gonadotropins on the molecular events controlling meiotic resumption of mouse oocytes.

Although studies suggest that the low competence of oocytes from prepubertal animals is due to their insufficient cytoplasmic maturation and that FSH improves oocyte maturation possibly by retarding meiotic progression and allowing more time for cytoplasmic maturation, the mechanisms by which puberty and gonadotropins regulate meiotic progression require additional detailed studies. For the first time, we observed that while meiotic progression was significantly slower, the maturation-promoting factor (MPF) activity of oocytes was significantly higher in prepubertal than in adult mice. To resolve this contradiction, we specified the molecules regulating the MPF activity and their localization during oocyte maturation in prepubertal and adult mice primed with or without gonadotropins. Our tests using corresponding enzyme regulators suggested that while activities of protein kinase A were unaffected, the activity of adenylate cyclase (ADCY) and phosphodiesterase increased while cell division cycle 2 homolog A (CDC2A) decreased significantly after puberty. While most of the adult oocytes had CDC2A protein concentrated in the germinal vesicle (GV) region, the majority of prepubertal oocytes showed no nuclear concentration of CDC2A. Maximally priming mice with equine chorionic gonadotropin brought the above parameters of prepubertal oocytes close to those in adult oocytes. Together, the results suggest that puberty and gonadotropin control oocyte meiotic progression mainly by regulating the ADCY activity and the concentration of the activated MPF toward the GV region.

[Circadian rhythms and systems biology]. / Biologie des systèmes et rythmes cellulaires.

Cellular rhythms represent a field of choice for studies in system biology. The examples of circadian rhythms and of the cell cycle show how the experimental and modeling approaches contribute to clarify the conditions in which periodic behavior spontaneously arises in regulatory networks at the cellular level. Circadian rhythms originate from intertwined positive and negative feedback loops controlling the expression of several clock genes. Models can be used to address the dynamical bases of physiological disorders related to dysfunctions of the mammalian circadian clock. The cell cycle is driven by a network of cyclin-dependent kinases (Cdks). Modeled in the form of four modules coupled through multiple regulatory interactions, the Cdk network operates in an oscillatory manner in the presence of sufficient amounts of growth factor. For circadian rhythms and the cell cycle, as for other recently observed cellular rhythms, periodic behavior represents an emergent property of biological systems related to their regulatory structure.

Requirement for p34cdc2 kinase is restricted to mitosis in the mammalian cdc2 mutant FT210.

The mouse FT210 cell line is a temperature-sensitive cdc2 mutant. FT210 cells are found to arrest specifically in G2 phase and unlike many alleles of cdc2 and cdc28 mutants of yeasts, loss of p34cdc2 at the nonpermissive temperature has no apparent effect on cell cycle progression through the G1 and S phases of the division cycle. FT210 cells and the parent wild-type FM3A cell line each possess at least three distinct histone H1 kinases. H1 kinase activities in chromatography fractions were identified using a synthetic peptide substrate containing the consensus phosphorylation site of histone H1 and the kinase subunit compositions were determined immunochemically with antisera prepared against the "PSTAIR" peptide, the COOH-terminus of mammalian p34cdc2 and the human cyclins A and B1. The results show that p34cdc2 forms two separate complexes with cyclin A and with cyclin B1, both of which exhibit thermal lability at the non-permissive temperature in vitro and in vivo. A third H1 kinase with stable activity at the nonpermissive temperature is comprised of cyclin A and a cdc2-like 34-kD subunit, which is immunoreactive with anti-"PSTAIR" antiserum but is not recognized with antiserum specific for the COOH-terminus of p34cdc2. The cyclin A-associated kinases are active during S and G2 phases and earlier in the division cycle than the p34cdc2-cyclin B1 kinase. We show that mouse cells possess at least two cdc2-related gene products which form cell cycle regulated histone H1 kinases and we propose that the murine homolog of yeast p34cdc/CDC28 is essential only during the G2-to-M transition in FT210 cells.

A complex containing p34cdc2 and cyclin B phosphorylates the nuclear lamin and disassembles nuclei of clam oocytes in vitro.

Cell-free extracts prepared from activated clam oocytes contain factors which induce phosphorylation of the single 67-kD lamin (L67), disassemble clam oocyte nuclei, and cause chromosome condensation in vitro (Dessev, G., R. Palazzo, L. Rebhun, and R. Goldman. 1989. Dev. Biol. 131:469-504). To identify these factors, we have fractionated the oocyte extracts. The nuclear lamina disassembly (NLD) activity, together with a protein kinase activity specific for L67, appear as a single peak throughout a number of purification steps. This peak also contains p34cdc2, cyclin B, and histone H1-kinase activity, which are components of the M-phase promoting factor (MPF). The NLD/L67-kinase activity is depleted by exposure of this purified material to Sepharose conjugated to p13suc1, and is restored upon addition of a p34cdc2/p62 complex from HeLa cells. The latter complex phosphorylates L67 and induces NLD in the absence of other clam oocyte proteins. Our results suggest that a single protein kinase activity (p34cdc2-H1 kinase, identical with MPF) phosphorylates the lamin and is involved in the meiotic breakdown of the nuclear envelope in clam oocytes.

Okadaic acid mimics a nuclear component required for cyclin B-cdc2 kinase microinjection to drive starfish oocytes into M phase.

G2-arrested oocytes contain cdc2 kinase as an inactive cyclin B-cdc2 complex. When a small amount of highly purified and active cdc2 kinase, prepared from starfish oocytes at first meiotic metaphase, is microinjected into Xenopus oocytes, it induces activation of the inactive endogenous complex and, as a consequence, drives the recipient oocytes into M phase. In contrast, the microinjected kinase undergoes rapid inactivation in starfish oocytes, which remain arrested at G2. Endogenous cdc2 kinase becomes activated in both nucleated and enucleated starfish oocytes injected with cytoplasm taken from maturing oocytes at the time of nuclear envelope breakdown, but only cytoplasm taken from nucleated oocytes becomes able thereafter to release second recipient oocytes from G2 arrest, and thus contains M phase-promoting factor (MPF) activity. Both nucleated and enucleated starfish oocytes produce MPF activity when type 2A phosphatase is blocked by okadaic acid. If type 2A phosphatase is only partially inhibited, neither nucleated nor enucleated oocytes produce MPF activity, although both do so if purified cdc2 kinase is subsequently injected as a primer to activate the endogenous kinase. The nucleus of starfish oocytes contains an inhibitor of type 2A phosphatase, but neither active nor inactive cdc2 kinase. Microinjection of the content of a nucleus into the cytoplasm of G2-arrested starfish oocytes activates endogenous cdc2 kinase, produces MPF activity, and drives the recipient oocytes into M phase. Together, these results show that the MPF amplification loop is controlled, both positively and negatively, by cdc2 kinase and type 2A phosphatase, respectively. Activation of the MPF amplification loop in starfish requires a nuclear component to inhibit type 2A phosphatase in cytoplasm.

A model for the adjustment of the mitotic clock by cyclin and MPF levels.

A mathematical model of cell cycle progression is presented, which integrates recent biochemical information on the interaction of the maturation promotion factor (MPF) and cyclin. The model retrieves the dynamics observed in early embryos and explains how multiple cycles of MPF activity can be produced and how the internal clock that determines durations and number of cycles can be adjusted by modulating the rate of change in MPF or cyclin concentrations. Experiments are suggested for verifying the role of MPF activity in determining the length of the somatic cell cycle.

The cell cycle then and now.

In the last few years a general model of cell cycle control has been established for all eukaryotic cells. Experiments from a variety of organisms and from a variety of experimental approaches have identified a protein kinase and its unstable regulatory subunit as the activator of mitosis; related molecules seem to be involved in the activation of chromosome replication. The identification of the biochemical components of these important regulatory pathways is providing several new insights into homeostatic and developmental control mechanisms in higher organisms.

Mos and the cell cycle: the molecular basis of the transformed phenotype.

The product of the mos proto-oncogene is a serine/threonine kinase that is expressed at high levels in germ cells. Mos is a regulator of meiotic maturation, and is required for the initiation and progression of oocyte meiotic maturation that leads to the production of unfertilized eggs. Mos is also a component of cytostatic factor, an activity that is believed to arrest oocyte maturation at meiotic metaphase II. There is evidence showing that the Mos protein is associated with tubulin in unfertilized eggs and transformed cells, raising the possibility that it is involved in the microtubular reorganization that occurs during M-phase. Inappropriate expression of its M-phase activity during interphase of the cell cycle may be responsible for its transforming activity.

Meiotic induction by Xenopus cyclin B is accelerated by coexpression with mosXe.

We have investigated the relationship between Xenopus laevis c-mos (mosXe) and the cyclin B component of maturation-promoting factor. Microinjection of Xenopus oocytes with in vitro-synthesized RNAs encoding Xenopus cyclin B1 or cyclin B2 induces the progression of meiosis, characterized by germinal vesicle breakdown (GVBD). By preinjecting oocytes with a mosXe-specific antisense oligonucleotide, we show that GVBD induced by cyclin B does not require expression of the mosXe protein. GVBD induced by cyclin B proceeds significantly faster than GVBD induced by progesterone or MosXe. However, coinjection of RNAs encoding cyclin B1 or cyclin B2 with mosXe RNA results in a 2.5- to 3-fold acceleration in GVBD relative to that induced by cyclin B alone. This acceleration of GVBD does not correlate with changes in the level of cyclin B1 and cyclin B2 phosphorylation.

Evidence for a role of protein kinase C zeta subspecies in maturation of Xenopus laevis oocytes.

A number of studies have demonstrated the activation of phospholipase C-mediated hydrolysis of phosphatidylcholine (PC-PLC) both by growth factors and by the product of the ras oncogene, p21ras. Evidence has been presented indicating that the stimulation of this phospholipid degradative pathway is sufficient to activate mitogenesis in fibroblasts as well as that it is sufficient and necessary for induction of maturation in Xenopus laevis oocytes. However, the mechanism whereby PC-PLC transduces mitogenic signals triggered by growth factors or oncogenes remains to be elucidated. In this study, data are presented that show the involvement of protein kinase C zeta subspecies in the channelling of the mitogenic signal activated by insulin-p21ras-PC-PLC in Xenopus oocytes as well as the lack of a critical role of protein kinase C isotypes alpha, beta, gamma, delta, and epsilon in these pathways.

Induction of starfish oocyte maturation by the beta gamma subunit of starfish G protein and possible existence of the subsequent effector in cytoplasm.

beta gamma subunits of G proteins were purified from starfish oocytes, and their role in the induction of oocyte maturation by 1-methyladenine was investigated. When injected into starfish oocytes, the purified beta gamma subunit of the starfish G protein induced germinal vesicle breakdown (GVBD) faster than that of bovine brain G protein. Injection of the starfish beta gamma into cytoplasm near the germinal vesicle (GV) induced GVBD earlier than when injected into the GV or the cytoplasm near the plasma membrane. Fluorescent-labeled beta gamma was retained in the injected area even after GVBD. Injected beta gamma also induced the formation of maturation-promoting factor as well as an increase of histone H1 kinase activity. These results suggest that beta gamma dissociates from alpha-subunit by the stimulation of 1-methyladenine and interacts with a cytoplasmic effector, which results in formation of active cdc2 kinase.

cdc25+ encodes a protein phosphatase that dephosphorylates p34cdc2.

To determine how the human cdc25 gene product acts to regulate p34cdc2 at the G2 to M transition, we have overproduced the full-length protein (cdc25Hs) as well as several deletion mutants in bacteria as glutathione-S-transferase fusion proteins. The wild-type cdc25Hs gene product was synthesized as an 80-kDa fusion protein (p80GST-cdc25) and was judged to be functional by several criteria: recombinant p80GST-cdc25 induced meiotic maturation of Xenopus oocytes in the presence of cycloheximide; p80GST-cdc25 activated histone H1 kinase activity upon addition to extracts prepared from Xenopus oocytes; p80GST-cdc25 activated p34cdc2/cyclin B complexes (prematuration promoting factor) in immune complex kinase assays performed in vitro; p80GST-cdc25 stimulated the tyrosine dephosphorylation of p34cdc2/cyclin complexes isolated from Xenopus oocyte extracts as well as from overproducing insect cells; and p80GST-cdc25 hydrolyzed p-nitrophenylphosphate. In addition, deletion analysis defined a functional domain residing within the carboxy-terminus of the cdc25Hs protein. Taken together, these results suggest that the cdc25Hs protein is itself a phosphatase and that it may function directly in the tyrosine dephosphorylation and activation of p34cdc2 at the G2 to M transition.

Mitosis-promoting factor activity of inducer mitotic cells may affect radiation yield of interphase chromosome breaks in the premature chromosome condensation assay.

We measured mitosis-promoting factor (MPF) activity in two cell lines, CHO and HeLa, extensively used at mitosis as inducers in the assay of premature chromosome condensation to study the yield and the repair kinetics of radiation damage in interphase chromosomes of diverse cell lines. We found a 2.5-fold higher MPF activity in HeLa as compared to CHO mitotic cells per mg of crude extract protein. HeLa mitotic cells, when used as inducers of premature chromosome condensation, uncovered two times more interphase chromosome breaks in irradiated, nonstimulated human lymphocytes as compared to CHO mitotic cells. A 2-fold increase in the yield of interphase chromosome breaks with HeLa mitotics was also observed in G1 cells from plateau-phase CHO cultures. Thus, MPF activity may be a contributing factor of the process that transforms radiation-induced DNA damage to chromosome breaks, and subsequently to other types of lethal chromosome aberrations. We speculate that the level and the control in the cell cycle of MPF activity may influence the radiosensitivity of cells to killing. The results strongly suggest that a direct comparison between the yields of interphase chromosome breaks measured in different laboratories may not be possible unless similar inducer cells with similar MPF activity are used.

Inhibition of c-mos protein kinase blocks mouse zygotes at the pronuclei stage.

The c-mos gene product is required for activation of the maturation-promoting factor (MPF) during oocyte maturation. The c-mos protein also acts as a cytostatic factor which is responsible for meiotic metaphase arrest of vertebrate eggs via stabilization of MPF. Here we show that mouse zygotes contain the c-mos protein. Introduction of a kinase-inhibitory anti-mos antibody into mouse zygotes 12 h after fertilization prevented the first cleavage of zygotes at the pronuclei stage. A second anti-mos antibody, known to allow the mos kinase to function, did not interfere with the formation of two-cell embryos. In addition to its known role in MPF activation in oocyte maturation and meiotic metaphase arrest, our findings indicate that the c-mos protein kinase is also required for completion of pronuclei breakdown following fertilization of mouse eggs.

Requirement of the c-mos protein kinase for murine meiotic maturation.

Anti-sense mos oligonucleotides have been shown to block steps required for meiotic maturation of oocytes. In Xenopus oocytes, the block prevents germinal vesicle breakdown (GVBD), an early step in oocyte maturation. In mice the block induced by anti-sense mos occurs at a later step in oocyte maturation concerned with the first polar body emission. Here, we show that mouse oocyte maturation is blocked by introduction of anti-mos antibodies into immature functional oocytes. Antibodies that inhibit the mos kinase blocked GVBD. In contrast, anti-mos antibodies that permit the mos kinase to function do not block GVBD, but interfere with polar body formation. Antibodies pre-reacted with excess cognate peptide had no observable effects on maturation. These results indicate that the c-mos kinase function is required for activation of maturation-promoting factor (MPF) during meiosis. These findings are also consistent with our previous studies which show that the mos kinase directly phosphorylates cyclin B2, a component of MPF (Roy et al., 1990).