Immobilized methylglyoxal-bis(guanylhydrazone) induces starfish oocyte maturation

Methylglyoxal-bis(guanylhydrazone) diHCl (MGBG), an inhibitor of S-adenosylmethionine decarboxylase, was found to induce starfish oocyte maturation at concentrations above 30 microM. Among several analogs of MGBG three induce oocyte maturation and one lacks the maturation-inducing activity while possessing the S-adenosylmethionine decarboxylase-inhibiting activity. Although MGBG is required during a slightly longer period than the natural hormone 1-methyladenine (1-MeAde), the maturation kinetics are identical. MGBG-induced maturation is sensitive to the same inhibitors as 1-MeAde-induced maturation (theophylline, caffeine, procaine, nicotine, NH4Cl, dansylcadaverine, vinblastine, R24571, and trifluoperazine). Inhibition is reversed by increasing the MGBG concentration. MGBG also induces an increase of protein phosphorylation. MGBG and 1-MeAde were separated on the basis of charcoal adsorption, MgSO4 precipitation, and thin-layer chromatography. MGBG covalently linked to CH-Sepharose 4B induces maturation in oocytes whose jelly layer and vitelline coat have been removed by a moderate pronase treatment, but not in the untreated oocytes. The MGBG-CH-Sepharose 4B beads come in close contact with the plasma membrane only in the pronase-treated oocytes. The mode of action of MGBG and the implications of these results in the purification of the 1-MeAde receptor are discussed.     

Isolation and characterization of a novel 530-kDa protein complex (PC530) capable of associating with the 20S proteasome from starfish oocytes

A novel protein complex called PC530 was purified concomitantly with proteasomes from oocytes of the starfish, Asterina pectinifera, by chromatography with DEAE-cellulose, phosphocellulose, Mono Q, and Superose 6 columns. The molecular mass of this complex was estimated to be 530 kDa by Ferguson plot analysis and about 500 kDa by Superose 6 gel filtration. Since the 1500-kDa proteasome fractions contain the PC530 subunits as well as the 20S proteasomal subunits, and also since the purified PC530 and the 20S proteasome were cross-linked with a bifunctional cross-linking reagent, it is thought that PC530 is able to associate with the 20S proteasome. The PC530 comprises six main subunits with molecular masses of 105, 70, 50, 34, 30, and 23 kDa. The 70-kDa subunit showed a sequence similarity to the S3/p58/Sun2/Rpn3p subunit of the 26S proteasome, whereas the other subunits showed little or no appreciable similarity to the mammalian and yeast regulatory subunits. These results indicate that starfish oocytes contain a novel 530-kDa protein complex capable of associating with the 20S proteasome, which is distinctly different from PA700 or the 19S regulatory complex in molecular size and subunit composition.     

Participation of proteasome-associating complex PC500 in starfish oocyte maturation as revealed by monoclonal antibodies

We previously reported that immature starfish oocytes contain a novel 530-kDa proteasome-associating complex PC500 [previously named PC530; E. Tanaka, M. Takagi Sawada, C. Morinaga, H. Yokosawa, H. Sawada, Isolation and characterization of a novel 530-kDa protein complex (PC 530) capable of associating with the 20S proteasome from star fish oocytes, Arch. Biochem. Biophys. 374 (2000) 181-188]. In the present study, in order to obtain an insight into the biological function of this complex, we investigated the effects of anti-PC500 monoclonal antibodies on oocyte maturation of the starfish Asterina pectinifera. A monoclonal antibody 7C5 strongly inhibited germinal vesicle breakdown (GVBD) in a concentration-dependent manner. In contrast to the inhibitory effect of the 7C5 antibody on GVBD, no inhibition of egg cleavage was observed in a 7C5-antibody-microinjected single blastomere in a 2-cell stage embryo. These results indicate that PC500 plays a key role in starfish oocyte maturation in a meiosis-specific manner.     

Enzymatic properties of the proteasome purified from starfish oocytes and its catalytic subunits involved in oocyte maturation

The 20S proteasome was purified from oocytes of the starfish Asterina pectinifera and its enzymatic properties were investigated. The chymotrypsin-like activities were potently inhibited by PSI as well as MG115, whereas the trypsin-like and peptidyl-glutamyl peptide-hydrolyzing (PGPH) activities were not or only weakly inhibited by PSI and MG115. The inhibitory ability of MG115 toward germinal vesicle breakdown (GVBD) coincided with those toward the trypsin-like and PGPH activities, and PSI showed no inhibitory effect on GVBD. We have previously reported that the inhibition pattern toward GVBD of peptidyl-argininals, which potently inhibited the proteasomal trypsin-like activity rather than the chymotrypsin-like activity, correlated with the inhibition pattern toward the chymotrypsin-like activity of the proteasome. These results, together with the peptidyl-argininals scarcely inhibiting the PGPH activity at concentrations sufficient for the inhibition toward GVBD, indicate that both the chymotrypsin-like and trypsin-like activities, but not the PGPH activity, of the proteasome are responsible for degradation of the physiological substrate during starfish oocyte maturation. It was also suggested that the inhibition of a single catalytic site of the proteasome is not sufficient for prevention of the proteasomal function.     

Stereospecific induction of starfish oocyte maturation by (8R)-hydroxyeicosatetraenoic acid

Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine. This induction of meiotic divisions can be triggered also by four fatty acids: 5,8,11-20:3; 5,8,11,14-20:4 (arachidonic acid); 6,9,12,15-20:4; 5,8,11,14,17-20:5, all other fatty acids being completely inactive. This maturation triggered by eicosanoids occurs in the micromolar range and is facilitated by the presence of calcium. A variety of arachidonic acid derivatives (esters, epoxides, etc.) and metabolites (cyclooxygenase and lipoxygenase products) has been tested; the biological activity is restricted to 8-hydroxyeicosatetraenoic acid (8-HETE), other mono- and poly-HETEs being completely inactive. Maturation triggered by 8-HETE occurs around 10 nM and is insensitive to the presence of calcium. 8-HETE methyl ester and 8-hydroperoxyeicosatetraenoic acid are able to induce maturation at higher concentrations. Both (8S) and (8R) stereoisomers have been tested; the biological activity is strictly restricted to the (8R) isomer. 8-HETE triggers a complete maturation, i.e. maturation-promoting factor appearance, germinal vesicle breakdown, emission of the polar bodies, and formation of a female pronucleus. (8R)-HETE, but not (8S)-HETE, triggers the typical decrease in cyclic AMP concentration induced by 1-methyladenine and the burst of protein phosphorylation associated with maturation. Starfish oocytes oxidize exogenous arachidonic acid into 8-HETE and other HETEs. 8-HETE was identified, after high pressure liquid chromatography purification, by gas chromatography mass spectrometry. Furthermore, it was found that the starfish oocytes only produce the (8R)-HETE isomer. This highly stereospecific induction of oocyte maturation by (8R)-HETE suggests that this fatty acid, or a very closely related fatty acid, may play a role in the transduction of the 1-methyladenine message at the plasma membrane level.     

A self-compartmentalizing protease in Rhodococcus: the 20S proteasome

The 26S proteasome represents a major, energy-dependent and self- compartmentalizing protease system in eukaryotes. The proteolytic core of this complex, the 20S proteasome, is also ubiquitous in archaea. Although absent from most eubacteria, this multi- subunit protease was recently discovered in Rhodococcus and appears to be confined to actinomycetes. The eubacterial 20S proteasome represents an attractive complementary system to study proteasome assembly, quaternary structure, and catalytic mechanism. In addition, it is likely to contribute substantially to our understanding of the role of various self-compartmentalizing proteases in bacterial cells.     

Induction of starfish oocyte maturation by disulfide-reducing agents

Oocyte maturation was found to be induced by disulfide-reducing agents such as dithiothreitol (DTT) and 2,3-dimercapto-1-propanol (BAL) in the starfish, Asterina pectinifera. The follicular envelopes around the oocytes broke and retracted into small clumps of cells on treatment with these reagents, as in the case of 1-methyladenine. Upon insemination, fertilizable eggs obtained by treatment with DTT formed a tight fertilization membrane and underwent cleavage. Such eggs developed normally to bipinnaria larvae. Cysteine and glutathione-SH had no effect in inducing oocyte maturation. On the other hand, pretreatment with sulfhydryl reagents such as p-chloromercurybenzoate (PCMB), iodoacetamide (IAM) and N-ethylmaleimide (NEM) completely suppressed 1-methyladenine-induced oocyte maturation. This inhibitory effect of sulfhydryl reagents on oocyte maturation was diminished by subsequent treatment with DTT or BAL with or without 1-methyladenine. Pretreatment with o-iodosobenzoate failed to inhibit 1-methyladenine-induced oocyte maturation.     

Development of calcium release mechanisms during starfish oocyte maturation

In response to the maturation-inducing hormone 1-methyladenine, starfish oocytes acquire increased sensitivity to sperm and inositol trisphosphate (InsP3), stimuli that cause a release of calcium from intracellular stores and a rise in intracellular free calcium. In the immature oocyte, the calcium release in response to 10 sperm entries is less than that seen with a single sperm entry in the mature egg. Likewise, the sensitivity to injected InsP3 is less in the immature oocyte. Approximately 100 times as much InsP3 is required to obtain the same calcium release in an immature oocyte as in a mature egg. However, with saturating amounts of InsP3, immature oocytes and mature eggs release comparable amounts of calcium. These results indicate that although calcium stores are well-developed in the immature oocyte, mechanisms for releasing the calcium develop fully only during oocyte maturation.     

Oocyte-surface factor responsible for 1-methyladenine-lnduced oocyte maturation in starfish

Fully grown oocytes of the starfish Asterina pectinifera, undergo breakdown of their germinal vesicles and subsequent maturation on treatment with 1-methyladenine (1-MeAde). However, oocytes treated with seawater containing 0.010% Triton X-100 lost the capacity to respond to 1-MeAde and their germinal vesicles remained intact. These decapacitated oocytes once again ac-quired the capacity to respond to l-Me Ade when they were incubated in sea water containing the extract of fully grown oocytes treated with Triton X-100, from which the Triton X-100 was removed after extraction by means of Bio-Beads SM-2 (TXE). Recovery of the capacity was also observed after washing such TXE-treated oocytes with sea water. These results suggest that some factor (probably 1-MeAde receptor or its fragment), extracted from the oocyte surface (plasma mem-brane) by nonionic detergent, was reconstituted on the oocyte surface so that the capacity of the oocytes to respond to 1-MeAde was recovered. The factor was heat-stable and resistant to treat-ment with proteolytic enzymes.     

Comparative proteome analysis of changes in the 26S proteasome during oocyte maturation in goldfish

Proteasomes are large, multi-subunit particles that act as the proteolytic machinery for most of the regulated intracellular protein degradation in eukaryotic cells. An alteration of proteasome function may be important for the regulation of the meiotic cell cycle. To study the change at the subunit level of the 26S proteasome during meiotic maturation, we purified 26S proteasomes from immature and mature oocytes of goldfish. Two-dimensional polyacrylamide gel electrophoresis was used to separate proteins. For differential analysis, whole spots of the 26S proteasome from goldfish oocytes were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and database analysis. Four spots that were different (only detected in mature oocyte 265 proteasomes and not in immature ones) and four protein spots that were up- or down-regulated were identified unambiguously. The mature-specific spots were not 26S proteasome components but rather their interacting proteins, and were identified as chaperonin-containing TCP-1 subunits and myosin light chain. Minor spots of three subunits of the 20S core particle and one of the 19S regulatory particle showed meiotic cell cycle-dependent changes. These results demonstrate that modifications of proteasomal subunits and cell cycle phase-dependent interactions of proteins with proteasomes occur during oocyte maturation in goldfish.     

Protein phosphorylation and oocyte maturation. I. Induction of starfish oocyte maturation by intracellular microinjection of a phosphatase inhibitor, alpha-naphthylphosphate

Oocyte maturation (meiosis re-initiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Following hormonal stimulation of the oocyte, an intracellular Maturation Promoting Factor (MPF) appears in the cytoplasm which triggers nuclear envelope breakdown and maturation divisions. alpha-Naphthylphosphate (alpha-NP), a widely used phosphatase inhibitor/substrate, was found to induce oocyte maturation when microinjected intracellularly (50% maturation of 3.5 mM; 100% above 6mM, final intracellular concentration) into oocytes of Marthasterias and Asterias but not of Astropecten. As 1-MeAde, alpha-NP triggers a complete maturation, i.e. germinal vesicle breakdown, extrusion of the two polar bodies and formation of the female pronucleus. The kinetics of alpha-NP-induced maturation (35-45 min) is, however, longer than the kinetics of 1-MeAde-induced maturation (18-20 min). The addition of alpha-NP externally to oocytes does not trigger maturation. Among several reported phosphatase inhibitors, including two natural protein phosphatase inhibitors and several products structurally related to alpha-NP, only alpha-NP was found capable of inducing maturation when microinjection into oocytes. alpha-NP triggers the appearance of MPF activity in the cytoplasm of oocytes into which it has been injected. Although alpha-NP-induced maturation is insensitive to inhibitors whose action is known to be restricted to the hormone-dependent period (such as the protease inhibitor leupeptin), it is blocked by inhibitors of MPF action (such as nicotinamide and lithium). Finally it was found that alpha-NP-induced maturation is inhibited by simultaneous microinjection of protein phosphatase-2A; also, alpha-NP, classically used as an inhibitor of acid and alkaline phosphatases, is able to inhibit protein phosphatases, is able to inhibit protein phosphatases 1 and 2 A. The addition of alpha-NP to oocytes increases the level of phosphorylated proteins. These results constitute direct evidence that an elevated level of phosphorylated proteins is sufficient to trigger MPF activity and to induce maturation.     

Disappearance of a novel protein component of the 26S proteasome during Xenopus oocyte maturation

We have prepared polyclonal antibodies against Xenopus 20S proteasomes. The antibodies cross-react with several proteins that are common to 20S and 26S proteasomes and with at least two proteins that are unique to 26S proteasomes. The antibodies were used to analyze changes in the components of proteasomes during oocyte maturation and early development of Xenopus laevis. A novel protein with a molecular weight of 48 kDa, p48, was clearly detected in immature oocytes, but was found at very low levels in mature oocytes and ovulated eggs. p48 was reduced to low levels during oocyte maturation, after maturation-promoting factor was activated. The amount of p48 in eggs remained low during early embryonic development, but increased again after the midblastula transition. These results show that at least one component of 26S proteasomes changes during oocyte maturation and early development and suggest that alterations in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycles, of the early embryo.     

Inhibition of starfish oocyte maturation by some inhibitors of proteolytic enzymes

Starfish oocyte maturation is triggered by a natural hormone, 1-methyladenine (1-MeAde), produced in the follicle cells, or artificially by dithiothreitol (DTT). These substances act on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), which induces germinal vesicle breakdown (GVBD) and subsequent processes of meiotic maturation. Further, MPF is amplified in immature oocytes that have received the injection of MPF. In this paper the effect of leupeptin and antipain, protease inhibitors of microbial origin, on starfish oocyte maturation was investigated. The protease inhibitors were found to inhibit 1-MeAde-induced maturation when they were applied externally or injected into oocytes. DTT-induced maturation was also inhibited by injection of leupeptin. However, leupeptin did not inhibit the maturation-inducing action of MPF or MPF amplification. These results show that the protease inhibitors suppress the production of MPF by 1-MeAde or DTT, suggesting that some endogenous protease(s) acts in the production of MPF.     

Synergistic actions of disulfide-reducing agents on 1-methyladenine-induced oocyte maturation in starfish

Several disulfide-reducing agents, such as dithiothreitol, 2,3-dimercapto-1-propanol, cysteine ethyl ester, and cysteine methyl ester enhanced the effectiveness of 1-methyladenine (1-MeAde) to induce oocyte maturation in the starfish Asterina pectinifera. This enhancement occurred at relatively low concentrations at which these agents by themselves were ineffective in inducing oocyte maturation. The agents caused a marked (about twofold) increase in specific [1-3H]MeAde binding. The binding increased directly in relation to the potency of the agents in enhancing 1-MeAde action. Scatchard analysis indicated that dithiothreitol increased the Bmax without affecting the affinity of 1-MeAde binding. These results strongly suggest that disulfide-reducing agents enhance the maturational action of 1-MeAde by increasing the number of 1-MeAde binding sites in oocyte cortices.     

Effect of L-phenylalanine on I-methyladenine production and spontaneous oocyte maturation in starfish

5-Bromodeoxyuridine (BUdR)-resistant cells were obtained from N-methyl-N′-nitro-N-nitrosoguanidine (NTG)-treated soybean protoplasts and cultured in liquid nutrient medium containing BUdR (20 μg/ml) and uridine (100 μg/ml). Addition of uridine to the medium improved growth of the BUdR-resistant cells. The growth of BUdR-resistant cells was partly inhibited when hypoxanthine, aminopterine, glycine and thymidine were added to the medium. Both BUdR-resistant and BUdR-sensitive cells exhibited thymidine kinase activity. CsCl density gradient analyses showed that the DNA of BUdR-resistant cells, which were cultured in the presence of BUdR, had a buoyant density of 1.703 g/ml, while the DNA of the parental soybean cells grown without BUdR had a buoyant density of 1.692 g/ml. Uptake of ³H-thymidine or ¹⁴C-BUdR by the cells occurred in both BUdR-resistant and BUdR-sensitive cells. CsCl density gradient patterns of labelled DNA also demonstrated that ¹⁴C-BUdR and ³H-thymidine were incorporated into the DNA of BUdR-resistant cells, as well as into that of BUdR-sensitive cells.     

Induction and inhibition of amphibian (Rana pipiens) oocyte maturation by protease inhibitor (TPCK)

We report for the first time that oocyte nuclear and cytoplasmic maturation are triggered in vitro in non-hormone-treated amphibian (Rana pipiens) ovarian follicles following transient exposure to synthetic chymotrypsin inhibitor Nα-tosyl-L-phenylalanine-chloromethyl ketone (TPCK). The mechanism of action of TPCK in regulating oocyte maturation was investigated and compared to that induced by progesterone or pituitary hormone. Follicular oocytes failed to mature following continuous exposure to the same doses of TPCK in the presence or absence of progesterone. Continuous treatment of follicles with lower levels of TPCK occasionally induced GVBD in the absence of progesterone and augmented maturational effects of low levels of progesterone. TPCK induced maturation of intrafollicular oocytes without stimulating progesterone production and also induced maturation of naked oocytes. Stimulation of follicular progesterone synthesis following gonadotropin stimulation or addition of pregnenolone was inhibited by TPCK, indicating that TPCK affects metabolic processes in both the somatic and germinal components of the ovarian follicle. Oocyte maturation induced by either TPCK or progesterone was inhibited by cycloheximide, calcium-deficient medium, and forskolin. Results suggest that TPCK induces oocyte maturation independent of steroidogenesis via mechanisms similar to those triggered by progesterone involving protein synthesis, formation of cytoplasmic maturation-promoting factor (MPF), and changes in cAMP levels. Our data indicate that a chymotrypsin-like protease plays a role(s) in regulating the oocyte meiotic maturation process.     

The proteasome maturation protein POMP facilitates major steps of 20S proteasome formation at the endoplasmic reticulum

The quality control of proteins mediated by the plasticity of the proteasome system is regulated by the timely and flexible formation of this multisubunit proteolytic enzyme complex. Adaptable biogenesis of the 20S proteasome core complex is therefore of vital importance for adjusting to changing proteolytic requirements. However, the molecular mechanism and the cellular sites of mammalian proteasome formation are still unresolved. By using precursor complex-specific antibodies, we now show that the main steps in 20S core complex formation take place at the endoplasmic reticulum (ER). Thereby, the proteasome maturation protein (POMP)--an essential factor of mammalian proteasome biogenesis--interacts with ER membranes, binds to alpha1-7 rings, recruits beta-subunits stepwise and mediates the association of mammalian precursor complexes with the ER. Thus, POMP facilitates the main steps in 20S core complex formation at the ER to coordinate the assembly process and to provide cells with freshly formed proteasomes at their site of function.     

Functional interaction of poly(ADP-ribose) with the 20S proteasome in vitro.

The nuclear enzyme poly(ADP-ribosyl) transferase (pADPRT) catalyzes the formation of poly(ADP-ribose) from NAD+. Several nuclear proteins and pADPRT itself are targets for the modification by poly(ADP-ribosyl)ation. It is demonstrated here that poly(ADP-ribose) or pADPRT automodified with poly(ADP-ribose) interacts noncovalently with the 20S proteasome in vitro. The interaction of pADPRT with the 20S proteasome requires the long ADP-ribose polymers formed by automodification of the pADPRT with poly(ADP-ribose). As a result pADPRT automodified with short ADP-ribose oligomers is unable to associate with the 20S proteasome. The interaction with poly(ADP-ribose) causes a specific stimulation of the peptidase activity of the 20S proteasome. Modified pADPRT does not serve as a substrate for the degradation by the 20S proteasome. No covalent modification of the 20S proteasome by ADP-ribosylation was observed. The results may point to a functional relationship between pADPRT and the 20S proteasome in a pathway protecting the cell from oxidative damage.