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.     

Arachidonic acid, 12- and 15-hydroxyeicosatetraenoic acids, eicosapentaenoic acid, and phospholipase A2 induce starfish oocyte maturation

In starfish oocyte maturation (meiosis reinitiation) is induced by the natural hormone 1-methyladenine (1-Me-Ade). This paper shows that arachidonic acid (AA) induces oocyte maturation at concentrations above 0.5 microM. This maturation shares many characteristics with 1-MeAde-induced maturation: same kinetics, same required contact time, same stimulations of protein phosphorylation and sodium influx. Although calcium facilitates the AA-induced but not the 1-MeAde-induced maturation, AA, like 1-MeAde, does not stimulate the uptake of calcium. Calcium does not facilitate the uptake of AA by oocytes. Out of 36 different fatty acids (saturated and unsaturated), only eicosatetraenoic (AA) and eicosapentaenoic acids were found to mimic 1-MeAde. Calcium-dependent phospholipases A2 from bee venom and Naja venom also induce maturation (0.1-1 unit/ml) when added externally to the oocytes. Phospholipase A2 inhibitors (quinacrine, bromophenacylbromide) block maturation; inhibition is reversed by increasing the 1-MeAde concentration and only occurs during the hormone-dependent period. AA is usually metabolized through oxidation by cyclooxygenase or lipoxygenase. Cyclooxygenase inhibitors (acetylsalicylic acid, indomethacin, tolazoline) do not block maturation; prostaglandins E2, D2, F2 alpha, I2, and thromboxane B2 do not induce meiosis reinitiation. On the other hand, lipoxygenase inhibitors (quercetin, butylated hydroxytoluene, and eicosatetraynoic acid) block 1-MeAde-induced maturation; although leukotrienes (A4, B4, C4, D4, E4) have no effects on oocytes, two other lipoxygenase products, 12- and 15-hydroxyeicosatetraenoic acids (and their corresponding hydroperoxy-) induce oocyte maturation (around 1 microM). The possible mode of action of the fatty acids inducing oocyte maturation is discussed.     

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.     

Metabolism of 12(R)-hydroxyeicosatetraenoic acid by rat liver microsomes

The in vitro metabolism of 12(R)-hydroxyeicosatetraenoic acid was studied using freshly isolated rat liver microsomes. Ten metabolites were isolated and identified by a combination of ultraviolet spectroscopy and gas chromatography/mass spectrometry. The two major metabolites were dihdroxyeicosatetraenoic acids generated by ω /ω − 1 hydroxylation. Oxidation at C-5 resulted in the formation of four leukotriene-like compounds, two of which differed from leukotriene B₄ in double-bond geometry alone. The other two differed from leukotriene B₄ in olefin geometry and C-5 configuration. Epoxidation at the 14,15-olefin resulted in the formation of two diastereomeric epoxy alcohols, while C-16 hydroxylation gave two diastereomeric dihydroxyeicosatetraenoic acids.     

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.     

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.     

Inhibition of starfish oocyte maturation by leupeptin analogs, potent trypsin inhibitors

Inhibition of subsite-substituted leupeptin analogs, potent trypsin inhibitors, on 1-methyladenine-induced germinal vesicle breakdown was investigated in a starfish, Asterina pectinifera. Of benzyloxycarbonyl(Z)-Leu-P2-argininals, the analog with Ser at P2 residue was the strongest inhibitor, and those with Pro, Leu greater than Thr greater than Gly were followed in this order. In Z-P3-Ser-argininals, ranking of the inhibitory ability was as follows: Phe greater than Leu much greater than Pro greater than Ala at P3 residue. Among 11 analogs synthesized, Z-Phe-Ser-argininal showed the strongest inhibition. The inhibitory potency of the analog was 100-fold stronger than that of leupeptin (acetyl-Leu-Leu-argininal). Thus, trypsin-like enzyme possessing a narrow subsite specificity participates in oocyte maturation in the starfish.     

Participation of 650-kDa protease (20 S proteasome) in starfish oocyte maturation.

A protease involved in oocyte maturation of a starfish, Asterina pectinifera, was explored. Trypsin-like and chymotrypsin-like activities of the 650-kDa protease in oocyte extract were revealed to increase more than twice under the influence of 1-methyladenine before germinal vesicle breakdown (GVBD) during maturation. The inhibitory potencies of leupeptin and its five analogs against the chymotrypsin-like activity, but not the trypsin-like activity, of this protease was well in accord with those against GVBD (Takagi Sawada et al. (1989). Dev. Biol. 133, 609-612). These results indicate that the chymotrypsin-like activity of the 650-kDa protease (most probably 20 S proteasome) plays a key role in starfish oocyte maturation.     

Extrafollicular mediation of oocyte maturation by radial nerve factor in starfish Pisaster ochraceus

In starfish ovaries follicle cells that envelop each oocyte are thought to mediate the production of a maturation inducing substance (MIS), identified as 1-methyladenine, that induces maturation and spawning of oocytes after exposure to a gonadotropic substance secreted by the radial nerve (RNF). Studies were carried out to assess the possible role of extrafollicular cells within the ovarian wall in mediating this signal transduction process in the ovary of Pisaster ochraceus. Oocyte maturation and spawning occurred following the addition of RNF to intact ovarian tissue in vitro whereas no maturation occurred following the addition of RNF to germinal vesicle (GV) oocytes or GV oocytes surrounded by follicle cells. In contrast, oocyte maturation occurred when small ovarian wall fragments, lacking mature follicles, were incubated with GV oocytes and RNF. Neither actinomycin D nor cycloheximide altered RNF induction of oocyte maturation in the presence of the ovarian wall tissue whereas preheating (boiling water for 5 min) the tissue obliterated its response to RNF. Non-ovarian tissues failed to produce MIS in response to RNF. Results suggest that ovarian components other than the follicle cells that envelop fully grown immature oocyte are responsive to RNF and represent a significant and previously unrecognised intra-ovarian source of MIS.     

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.     

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.     

6-Dimethylaminopurine blocks starfish oocyte maturation by inhibiting a relevant protein kinase activity

The puromycin analog N6,N6-dimethyladenine (6-dimethylaminopurine or 6-DMAP) was found to inhibit meiosis reinitiation in starfish oocytes stimulated by the natural hormone 1-methyladenine. Increasing concentrations of this agent delayed and eventually blocked germinal vesicle breakdown. They were found to be effective even when applied during the hormone-independent period, after the oocytes had been already committed to reinitiate meiosis. 6-DMAP mimics most of the effects of emetine since it induces protein dephosphorylation, inhibits polar body formation, and promotes the precocious appearance of resting nuclei. However, unlike emetine, 6-DMAP does not affect protein synthesis. The effect of this agent cannot be accounted for by a stimulation of the protease or phosphoprotein phosphatase activities since the rate and extent of protein dephosphorylation do not increase in its presence. Data from in vivo and in vitro endogenous protein phosphorylation experiments suggest rather that 6-DMAP may directly or indirectly affect the activity of a relevant c-AMP and Ca2+-independent protein kinase which is stimulated after hormone addition and seems to support starfish oocyte maturation.     

In vitro inhibition of oocyte and follicular maturation and spawning in starfish (Asterias forbesi) by 2-4-dinitrophenol

The in vitro effects of 2-4-dinitrophenol (DNP) on spawning and follicular and oocyte maturation in starfish ovaries and its various cellular components were investigated. Spawning and oocyte and follicular maturation induced by starfish gonadotropin radial nerve factor (RNF) in isolated ovarian fragments were all inhibited by appropriate doses of DNP. DNP inhibits processes which occur shortly after addition of the gonadotropin; in ovarian fragments insensitivity to DNP inhibition occurred shortly after addition of RNF but prior to initiation of spawning. Spontaneous follicular and oocyte maturation which occurred following release of ovarian follicles into sea water was prevented by DNP. In non-spontaneously maturing follicles released from the ovary, DNP inhibited both follicle and oocyte maturation induced by the secondary stimulator of spawning and maturation, 1-methyladenine (1-MA). DNP also inhibited 1-MA induced meiotic maturation in isolated immature oocytes incubated in the absence of follicle cells. Inhibition of oocyte maturation was not associated with inhibition of 3H-1-MA incorporation by isolated oocytes. Immature oocytes incubated in the presence of DNP underwent maturation following washing and subsequent exposure to 1-MA. Immature oocytes initially exposed to both 1-MA and DNP, however, showed decreased maturation responsiveness following washing and re-exposure to 1-MA. The results suggest that the inhibitory effects of DNP on spawning and oocyte maturation are the result of direct effects on the oocytes and possibly other cells and tissues within the ovary.     

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.     

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.     

SC-41930 inhibits neutrophil infiltration of the cavine dermis induced by 12(R)-hydroxyeicosatetraenoic acid

Psoriasis is a disease state characterized by epidermal proliferation, neutrophil infiltration, along with release of the proinflammatory mediators leukotriene-B4 (LTB4) and 12(R)-hydroxyeicosatetraenoic acid [12(R)-HETE]. LTB4 and 12(R)-HETE are chemoattractant to the neutrophil, the latter approximately 1000X less potent. LTB4 and 12(R)-HETE are present in psoriatic scale, the latter in quantities so much greater than LTB4 that it is proposed as a primary mediator of neutrophil infiltration in psoriasis. 12(R)-HETE, synthesized in optically pure form by a new, shorter route, was injected into the cavine dermis. At a dose of 25 micrograms m per intradermal site, 12(R)-HETE was a significant chemoattractant to the neutrophil (as assessed by dermal myeloperoxidase levels). SC-41930, 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]- 3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid, given intragastrically inhibited 12(R)-HETE-induced neutrophil infiltration of the cavine dermis with an ED50 value of 13.5 mg/kg. Compounds such as SC-41930 may well have utility for treating human psoriasis.     

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.