Translational regulation of cyclin B mRNA by 17alpha,20beta-dihydroxy-4-pregnen-3-one (maturation-inducing hormone) during oocyte maturation in a teleost fish, the goldfish (Carassius auratus)

17Alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP) was identified as maturation-inducing hormone (MIH) in several teleost fishes. In goldfish (Carassius auratus), 17alpha,20beta-DP induces oocyte maturation by stimulating the de novo synthesis of cyclin B, a regulatory subunit of maturation-promoting factor (MPF). In this study, we examined the control mechanisms of 17alpha,20beta-DP-induced de novo synthesis of cyclin B protein in oocytes, which is a prerequisite step for MPF activation during oocyte maturation in goldfish. Cycloheximide-treated oocytes failed to undergo meiotic maturation in response to 17alpha,20beta-DP; in this group neither cyclin B nor 34-kDa active cdc2 was detectable in oocytes. In contrast, oocytes exposed to actinomycin D plus 17alpha,20beta-DP or 17alpha,20beta-DP underwent maturation; in these groups both cyclin B and 34-kDa cdc2 were present. Northern blotting showed that cyclin B mRNA is present in both immature and mature oocytes. Sequence analysis revealed that goldfish cyclin B mRNA contains four copies of cytoplasmic polyadenylation element (CPE)-like motifs in the 3' noncoding region, suggesting that the initiation of cyclin B synthesis during oocyte maturation may be controlled by the elongation of poly (A) tail. We then examined the polyadenylation state of cyclin B mRNA during 17alpha,20beta-DP-induced oocyte maturation by means of a PCR poly (A) test, and found that cyclin B mRNA is polyadenylated during oocyte maturation. Polyadenylation of cyclin B mRNA occurred at the same time of germinal vesicle breakdown. Furthermore, cordycepin, an inhibitor of poly (A) addition of mRNA, prevented 17alpha,20beta-DP-induced oocyte maturation. These findings suggest that in goldfish oocytes, the synthesis of cyclin B protein is under translational control and that cytoplasmic 3' poly(A) elongation is involved in 17alpha,20beta-DP-induced translation of cyclin B mRNA.     

An inhibitor of p34cdc2/cyclin B that regulates the G2/M transition in Xenopus extracts.

The activity of maturation-promoting factor (MPF), a protein kinase complex composed of p34cdc2 and cyclin B, is undetectable during interphase but rises abruptly at the G2/M transition to induce mitosis. After the synthesis of cyclin B, the suppression of MPF activity before mitosis has been attributed to the phosphorylation of p34cdc2 on sites (threonine-14 and tyrosine-15) that inhibit its catalytic activity. We previously showed that the activity of the mitotic p34cdc2/cyclin B complex is rapidly suppressed when added to interphase Xenopus extracts that lack endogenous cyclin B. Here we show that a mutant of p34cdc2 that cannot be inhibited by phosphorylation (threonine-14-->alanine, tyrosine-15-->phenylalanine) is also susceptible to inactivation, demonstrating that inhibitory mechanisms independent of threonine-14 and tyrosine-15 phosphorylation must exist. We have partially characterized this inhibitory pathway as one involving a reversible binding inhibitor of p34cdc2/cyclin B that is tightly associated with cell membranes. Kinetic analysis suggests that this inhibitor, in conjunction with the kinases that mediate the inhibitory phosphorylations on p34cdc2, maintains the interphase state in Xenopus; it may play an important role in the exact timing of the G2/M transition.     

Cellular, biochemical and molecular mechanisms regulating oocyte maturation

The original model for regulation of oocyte maturation proposed by us in 1978 postulated that gap junction-mediated transmission of cAMP from the follicle cells to the oocyte inhibits meiosis and that luteinizing hormone (LH) terminates the flux of the follicle cAMP to the oocyte. A decrease in oocyte cAMP below inhibitory threshold occurs since oocytes lack the ability to generate sufficient amounts of cAMP to compensate for the phosphodiesterase activity. Our previous studies provided evidence to support this model. More recent studies in our laboratory were directed at identification of the cellular biochemical and molecular events initiated within rat oocytes upon the relief of cAMP inhibition. These studies: (i) identified an oocyte specific A kinase anchoring protein (AKAP) that is phosphorylated in oocytes resuming meiosis, (ii) confirmed that cdc25B governs meiosis reinitiation and demonstrated that its expression is translationally regulated, (iii) substantiated the indispensable role of proteasomal degradation at completion of the first meiotic division in a mammalian system, (iv) elucidated the role of MPF reactivation in suppressing interphase between the two meiotic divisions and (v) provided evidence that mos translation is negatively regulated by a protein kinase A (PKA)-mediated action of cAMP and is dependent on an active MPF. A detailed account on each of these findings is presented in this chapter.     

Evidence that Mos protein may not act directly on cyclin.

Using affinity-purified antiserum we have examined cyclin B2 levels in Xenopus oocytes at various stages of oogenesis. We found that cyclin B2 is detected from stage 2 to stage 6 as two bands, one of which is phosphorylated, and that cyclin B2 mass increases about 28-fold between stage 2 and stage 6. To examine the effect of Mos protein on cyclin phosphorylation, we microinjected synthetic Xenopus c-mos (c-mosxe) RNA into stages 4, 5, and 6 Xenopus oocytes. In stage 6 oocytes, maturation was induced by c-mosxe RNA, and, as is the case with progesterone treatment, all cyclin B2 was shifted to the phosphorylated form. However, c-mosxe RNA injected into stage 4 or 5 oocytes did not induce maturation or cause a shift in the relative proportion of the two cyclin B2 bands. These data suggest that Mos does not act directly to phosphorylate cyclin B2, causing the band shift during maturation. Cyclin B2 synthesis increases about 2-fold during maturation, in concert with total protein synthesis. Data from experiments on cyclin B2 stability indicate that the half-life of cyclin B2 is about 85 hr in stage 6. This suggests that if Mos protein has a direct effect on cyclin stability, it does so only at a later stage in oocyte maturation, but not at the onset.     

Multiple forms of maturation-promoting factor in unfertilized Xenopus eggs.

Maturation-promoting factor (MPF), which is functionally defined by its ability to induce frog oocyte maturation independent of protein synthesis, is hypothesized to be the mitotic inducer in eukaryotic cells. Previous studies have demonstrated that the cdc2 protein kinase complex (p34cdc2-cyclin) meets the criteria for MPF. In the present study, we show that MPF activity in extracts of unfertilized Xenopus eggs can be resolved into three fractions by Q-Sepharose chromatography. Of the total MPF activity recovered, approximately 20% was in the flow-through fraction that was accounted for by the cdc2 kinase complex, approximately 40% was in the 0.2 M NaCl eluate, and the remaining approximately 40% was in the 0.5 M NaCl eluate. Neither eluate contained cdc2 kinase, but each could activate cdc2 kinase upon microinjection into Xenopus oocytes. The MPF activity in the two eluates, but not in the flow-through fraction, could be depleted by the mitosis-specific monoclonal antibody MPM-2. This antibody has been shown to inhibit Xenopus oocyte maturation and deplete MPF activity from mature oocyte extract but does not recognize the cdc2 kinase complex. The three MPFs differed in apparent molecular size, H1 kinase activity, and stability at 4 degrees C. We propose that MPF activity in unfertilized Xenopus eggs resides in at least three different molecular species, the combined activities of which may be required for autoamplification of MPF.     

Mos/mitogen-activated protein kinase can induce early meiotic phenotypes in the absence of maturation-promoting factor: a novel system for analyzing spindle formation during meiosis I.

Mitogen-activated protein kinase (MAPK) is selectively activated by injecting either mos or MAPK kinase (mek) RNA into immature mouse oocytes maintained in the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). IBMX arrests oocyte maturation, but Mos (or MEK) overexpression overrides this block. Under these conditions, meiosis I is significantly prolonged, and MAPK becomes fully activated in the absence of p34cdc2 kinase or maturation-promoting factor. In these oocytes, large openings form in the germinal vesicle adjacent to condensing chromatin, and microtubule arrays, which stain for both MAPK and centrosomal proteins, nucleate from these regions. Maturation-promoting factor activation occurs later, concomitant with germinal vesicle breakdown, the contraction of the microtubule arrays into a precursor of the spindle, and the redistribution of the centrosomal proteins into the newly forming spindle poles. These studies define important new functions for the Mos/MAPK cascade in mouse oocyte maturation and, under these conditions, reveal novel detail of the early stages of oocyte meiosis I.     

Identification of mitosis-specific p65 dimer as a component of human M phase-promoting factor.

Antisera raised against two mitosis-specific protein kinases from human cells recognized a single 65-kDa polypeptide (p65) that is present in similar amounts in interphase and mitotic cell extracts. Immunoblot analysis of reduced and unreduced extracts revealed that p65 exists as a 65-kDa monomer during interphase but forms a 130-kDa disulfide-linked homodimer during mitosis. Several different antibodies recognizing the p34cdc2 protein kinase and cyclin B components of M phase-promoting factor (MPF) coprecipitated p65 from mitotic but not from interphase extracts. In addition, an anti-p65 immunoaffinity column substantially depleted mitotic extracts of histon H1 kinase activity assayed under conditions diagnostic for MPF. These results suggest that active human MPF may be a complex of p34cdc2, cyclin B, and dimeric p65. A sulfhydryl cycle, proposed in the earlier literature on the biochemistry of mitosis, might underlie the dimerization of p65 and formation of active MPF.     

The Mos/mitogen-activated protein kinase (MAPK) pathway regulates the size and degradation of the first polar body in maturing mouse oocytes.

Mos is an upstream activator of mitogen-activated protein kinase (MAPK) and, in mouse oocytes, is responsible for metaphase II arrest. This activity has been likened to its function in Xenopus oocytes as a component of cytostatic factor. Thus, Mos-deficient female mice (MOS-/-) are less fertile and oocytes derived from these animals fail to arrest at metaphase II and undergo parthenogenetic activation [Colledge, W. H., Carlton, M. B. L., Udy, C. B. & Evans, M. J. (1994) Nature (London) 370, 65-68 and Hashimoto, N., Watanabe, N., Furuta. Y., Tamemoto, B., Sagata, N., Yokoyama, M., Okazaki, K., Nagayoshi, M., Takeda, N., Ikawa, Y. & Aizawa, S. (1994) Nature (London) 370, 68-71]. Here we show that maturing MOS-/- oocytes fail to activate MAPK throughout meiosis, while p34cdc2 kinase activity is normal until late in metaphase II when it decreases prematurely. Phenotypically, the first meiotic division of MOS-/- oocytes frequently resembles mitotic cleavage or produces an abnormally large polar body. In these oocytes, the spindle shape is altered and the spindle fails to translocate to the cortex, leading to the establishment of an altered cleavage plane. Moreover, the first polar body persists instead of degrading and sometimes undergoes an additional cleavage, thereby providing conditions for parthenogenesis. These studies identify meiotic spindle formation and programmed degradation of the first polar body as new and important roles for the Mos/MAPK pathway.     

Mitogen-activated protein kinase activity in cumulus cells is essential for gonadotropin-induced oocyte meiotic resumption and cumulus expansion in the mouse

This study investigated the participation of MAPK in the resumption of meiosis [germinal vesicle breakdown (GVB)] in oocytes and cumulus expansion using oocyte-cumulus cell complexes (OCC) from Mos-null mice (Mos(tm1Ev)/Mos(tm1Ev), hereafter Mos(-/-)). MAPK activity was not detected in Mos(-/-) oocytes whether they matured in vivo or in vitro, with or without gonadotropin stimulation. Therefore, there are no pathways independent of MOS that activate MAPK during gonadotropin-induced maturation. In contrast, MAPK activity was always detected coincident with GVB in Mos(+/+) oocytes. Moreover, MAPK activity was detected in cumulus cells before gonadotropin-induced GVB in OCC regardless of genotype. A specific inhibitor (U0126) of MEK, a MAPKK required for MAPK activity, inhibited gonadotropin-induced GVB in OCC of both Mos(+/+) and Mos(-/-) mice. Activation of MAPK was downstream of elevation of cAMP. U0126 also inhibited cumulus expansion stimulated by FSH, epidermal growth factor, 8-bromo-cAMP, and recombinant growth differentiation factor-9. It is concluded that under the in vitro conditions used here, gonadotropin-induced GVB requires the participation of MAPK activity in the cumulus cells, but not in the oocyte. Moreover, the induction of cumulus expansion also requires the participation of MAPK, and this action is downstream of both elevation of cAMP and growth differentiation factor-9.     

Oncogenic ras triggers the activation of 42-kDa mitogen-activated protein kinase in extracts of quiescent Xenopus oocytes.

Quiescent, full-grown Xenopus oocytes, which are arrested at the G2/M border of meiosis, contain an inactive 42-kDa mitogen-activated protein kinase (p42MAPK) that is activated when oocytes are stimulated to resume the meiotic cell cycle. We have made extracts from these oocytes that respond to four cell cycle activators: oncogenic [Val12]Ras protein, clam cyclins A delta 60 and B delta 97, and the phosphatase inhibitor okadaic acid. All four induce the tyrosine phosphorylation and activation of p42MAPK. Both cyclins and okadaic acid, but not [Val12]Ras, also lead to activation of the endogenous cyclin B/cdc2 kinase complexes in extracts of quiescent oocytes. Using extracts prepared from cycloheximide-arrested interphase cells, we show that although p42MAPK activation can occur in response to cyclin-activated cdc2, the Ras-induced activation of p42MAPK occurs without intervening cdc2 activation. Neither the nononcogenic [Gly12]Ras nor [Val12,Arg186]Ras, a mutant that lacks the C-terminal consensus sequence directing prenylation and subsequent membrane association, is an effective activator of p42MAPK in vitro.     

Expression and modification of PKA and AKAPs during meiosis in rat oocytes

Meiosis in oocytes is initiated during fetal life, arrested around birth and resumed after puberty. Meiotic arrest is controlled by a cAMP-dependent protein kinase (PKA)-mediated cAMP action. We examined oocytes for the presence and modulation of the regulatory (R) subunits of PKA and the A-kinase anchoring proteins (AKAPs) that target PKA to specific subcellular locations. We found that rat oocytes express the two regulatory subunit isoforms, RI and RII of PKA. Immunocytochemistry revealed that the regulatory subunits underwent cellular translocation upon resumption of meiosis. We also demonstrated the presence of a novel 140 kDa AKAP, AKAP140 that exhibited a retarded electrophoretic motility at reinitiation of meiosis. The mobility shift of AKAP140 was susceptible to alkaline phosphatase and prevented by inhibition of p34cdc2 kinase. We conclude that rat oocytes express AKAP140 that is phosphorylated during meiosis. AKAP140 phosphorylation is sensitive to p34cdc2 kinase inhibitors. We hypothesize that AKAP140 and its phosphorylation state may influence the translocation of the R subunits of PKA throughout resumption of meiosis.     

Cell division in higher plants: a cdc2 gene, its 34-kDa product, and histone H1 kinase activity in pea.

The mitotic cell cycle of yeast and animal cells is regulated by the cdc2 gene and its product, the p34 protein kinase, and by other components of the MPF or histone H1 kinase complex. We present evidence that cdc2, p34, and a histone H1 kinase also exist in higher plants. Protein extracts from 10 plant species surveyed display a 34-kDa component recognized by a monoclonal antibody directed against an evolutionarily conserved epitope of fission yeast p34. Nondenatured protein extracts of mitotic Pisum sativum (garden pea) tissues were fractionated by gel filtration, electrophoretically separated under denaturing conditions, and immunoblotted. p34 crossreactive material was apparent in both low and high molecular mass fractions, indicating that pea p34 occurs as both a monomer and as part of a high molecular mass complex. Histone H1 kinase activity was found predominantly in the higher molecular mass fractions, those to which the least phosphorylated form of pea p34 was confined. We also report the cloning of the pea homologue of cdc2 by polymerase chain reaction. DNA sequence analysis reveals perfect conservation of the hallmark "PSTAIR" sequence motif found in all cdc2 gene products analyzed to date.     

Unscheduled cyclin B expression and p34 cdc2 activation in T lymphocytes from HIV-infected patients.

OBJECTIVE: To study the role of cell cycle regulation during HIV infection by investigating in vivo and in vitro cyclin B and p34 cdc kinase expression. METHODS: Cyclin B expression was analysed by Western blot in CD4 and CD8 cells from 25 HIV-infected patients and 24 uninfected individuals. In eight patients, a sequential analysis was performed after initiation of antiretroviral therapy (ART), and correlations with CD4 cell count and HIV viremia were studied. Sequential changes in cyclin B expression and p34 cdc kinase expression and activity were also studied in lymphocytes activated in vitro with phytohaemagglutinin (PHA). RESULTS: Lymphocytes from untreated HIV-infected patients demonstrate persistent in vivo overexpression of cyclin B in both CD4 and CD8 cell subpopulations. When cells are stimulated to proliferate in vitro, biochemical events that characterize the entrance into the cell cycle [ornithine decarboxylase (ODC) activity, interleukin 2 production, interleukin 2 alpha-chain receptor (IL-2R, CD25) expression, total protein synthesis, total DNA synthesis] show similar timing and sequence in lymphocytes from HIV-infected and uninfected individuals. However, in peripheral blood lymphocytes (PBL) from HIV-infected patients, cyclin B and p34 cdc kinase show premature expression during the cell cycle. Both in vivo cyclin B overexpression and in vitro unscheduled cyclin B expression were almost completely reversed 2-4 weeks after initiation of effective ART. CONCLUSION: Increased and unscheduled expression of cyclin B and p34 cdc kinase is consistently observed in CD4 and CD8 cells from HIV-infected patients, both in vivo and after in vitro mitogenic stimulation. These alterations correlate with the level of viremia and may provide a link between the perturbation of lymphocyte proliferative homeostasis and the exaggerated propensity towards apoptosis.     

Role of growth hormone and growth hormone receptor in oocyte maturation

A cytoplasmic activity in mature oocytes responsible for second meiotic metaphase arrest was identified over 30 years ago in amphibian oocytes. In Xenopus oocytes cytostatic factor (CSF) activity is initiated by the progesterone-dependent synthesis of Mos, a MAPK kinase kinase that activates the MAPK pathway. CSF arrest is mediated by a sole MAPK target, the protein kinase p90(Rsk). Rsk phosphorylates and activates the Bub1 protein kinase, which may cause metaphase arrest due to inhibition of the anaphase-promoting complex (APC) by a conserved mechanism defined genetically in yeast and mammalian cells. CSF arrest in vertebrate oocytes by p90(Rsk) provides a link between the MAPK pathway and the spindle assembly checkpoint in the cell cycle.     

Ca2+ triggers premature inactivation of the cdc2 protein kinase in permeabilized sea urchin embryos.

Exit from mitosis requires inactivation of the cyclin B-p34cdc2 protein kinase complex. Since increased cytosolic Ca2+ has been implicated as a potential trigger of mitotic progression, we directly tested the possibility that Ca2+ triggers the pathway responsible for inactivating the cdc2 kinase, using sea urchin embryos permeabilized at various stages of the cell cycle. In cells permeabilized during late interphase and prophase, micromolar Ca2+ induced premature inactivation of the cdc2 kinase without affecting the absolute amount of p34cdc2 protein. Inactivation was selective for the cdc2 kinase, as elevated Ca2+ had no effect on cAMP-dependent protein kinase activity. Premature cdc2 kinase inactivation did not require cyclin B destruction, but did coincide with the dissociation of cyclin B-p34cdc2 complexes. In cells permeabilized during prometaphase and metaphase, cdc2 kinase inactivation was Ca(2+)-independent, presumably because at these later times the inactivating pathway had been enabled prior to permeabilization. This work provides evidence that Ca2+ is the physiological trigger enabling cdc2 kinase inactivation during mitosis.     

Phosphorylation of the regulatory subunit of type II beta cAMP-dependent protein kinase by cyclin B/p34cdc2 kinase impairs its binding to microtubule-associated protein 2.

Subcellular localization of type II cAMP-dependent protein kinase is determined by the interactions of the regulatory subunit (RII) with specific RII-anchoring proteins. By using truncated NH2-terminal RII beta fusion proteins expressed in Escherichia coli and the mitotic protein kinase p34cdc2 isolated from HeLa cells or starfish oocytes, we investigated the in vitro phosphorylation of RII beta by these kinases. The putative site for phosphorylation by the mitotic kinases is Thr-69 in the NH2-terminal domain of RII beta. This phosphorylation site matches the consensus sequence X(T/S)PX(K/R) for p34cdc2 recognition and belongs to a well-conserved sequence found in all RII beta sequences known to date. In contrast to phosphorylation by casein kinase II or the cAMP-dependent protein kinase catalytic subunit, phosphorylation of RII beta by mitotic kinases impaired its interaction with a well-known RII-anchoring protein, the neuronal microtubule-associated protein 2. The potential regulatory significance of the phosphorylation of this site on the interaction with microtubule-associated protein 2 and other RII-anchoring proteins and the physiological relevance of this cyclin B/p34cdc2 kinase-catalyzed modification of RII beta (or phosphorylation by other proline-directed protein kinases) are discussed.     

p107wee1 is a dual-specificity kinase that phosphorylates p34cdc2 on tyrosine 15.

p107wee1 is a protein kinase that functions as a dose-dependent inhibitor of mitosis through its interactions with p34cdc2 in Schizosaccharomyces pombe. To characterize the kinase activity of p107wee1, its carboxyl-terminal catalytic domain was purified to homogeneity from overproducing insect cells. The apparent molecular mass of the purified protein (p37wee1KD) was determined to be approximately 37 kDa by gel filtration, consistent with it being a monomer. Serine and tyrosine kinase activities cofiltered with p37wee1KD, demonstrating that p107wee1 is a dual-specificity kinase. In vitro, p107wee1 phosphorylated p34cdc2 on Tyr-15 only when p34cdc2 was complexed with cyclin. Neither monomeric p34cdc2 nor a peptide containing Tyr-15 was able to substitute for the p34cdc2/cyclin complex in this assay. Furthermore, the phosphorylation of p34cdc2 by p107wee1 in vitro inhibited the histone H1 kinase activity of p34cdc2. These results indicate that p107wee1 functions as a mitotic inhibitor by directly phosphorylating p34cdc2 on Tyr-15 and that the preferred substrate for phosphorylation is the p34cdc2/cyclin complex.     

G2 arrest in Xenopus oocytes depends on phosphorylation of cdc25 by protein kinase A

Xenopus oocytes, which are arrested in G(2) of meiosis I, contain complexes of cyclin B-cdc2 (M phase-promoting factor) that are kept repressed by inhibitory phosphorylations on cdc2 at Thr-14 and Tyr-15. Progesterone induces a cytoplasmic signaling pathway that leads to activation of cdc25, the phosphatase that removes these phosphorylations, catalyzing entry into M phase. It has been known for 25 years that high levels of cAMP and protein kinase A (PKA) are required to maintain the G(2) arrest and that a drop in PKA activity is required for M phase-promoting factor activation, but no physiological targets of PKA have been identified. We present evidence that cdc25 is a critical target of PKA. (i) In vitro, cdc25 Ser-287 serves as a major site of phosphorylation by PKA, resulting in sequestration by 14-3-3. (ii) Endogenous cdc25 is phosphorylated on Ser-287 in oocytes and dephosphorylated in response to progesterone just before cdc2 dephosphorylation and M-phase entry. (iii) High PKA activity maintains phosphorylation of Ser-287 in vivo, whereas inhibition of PKA by its heat-stable inhibitor (PKI) induces dephosphorylation of Ser-287. (iv) Overexpression of mutant cdc25 (S287A) bypasses the ability of PKA to maintain oocytes in G(2) arrest. These findings argue that cdc25 is a physiologically relevant target of PKA in oocytes. In the early embryonic cell cycles, Ser-287 is phosphorylated during interphase and dephosphorylated just before cdc2 activation and mitotic entry. Thus, in addition to its role in checkpoint arrest, cdc25 Ser-287 serves as a site for regulation during normal, unperturbed cell cycles.