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.     

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.     

XGef is a CPEB-interacting protein involved in Xenopus oocyte maturation

XGef was isolated in a screen for proteins interacting with CPEB, a regulator of mRNA translation in early Xenopus development. XGef is a Rho-family guanine nucleotide exchange factor and activates Cdc42 in mammalian cells. Endogenous XGef (58 kDa) interacts with recombinant CPEB, and recombinant XGef interacts with endogenous CPEB in Xenopus oocytes. Injection of XGef antibodies into stage VI Xenopus oocytes blocks progesterone-induced oocyte maturation and prevents the polyadenylation and translation of c-mos mRNA; injection of XGef rescues these events. Overexpression of XGef in oocytes accelerates progesterone-induced oocyte maturation and the polyadenylation and translation of c-mos mRNA. Overexpression of a nucleotide exchange deficient version of XGef, which retains the ability to interact with CPEB, no longer accelerates oocyte maturation or Mos synthesis, suggesting that XGef exchange factor activity is required for the influence of overexpressed XGef on oocyte maturation. XGef overexpression continues to accelerate c-mos polyadenylation in the absence of Mos protein, but does not stimulate MAPK phosphorylation, MPF activation, or oocyte maturation, indicating that XGef may function through the Mos pathway to influence oocyte maturation. These results suggest that XGef may be an early acting component of the progesterone-induced oocyte maturation pathway.     

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.     

XCdh1 is involved in progesterone-induced oocyte maturation

The molecular events triggered during progesterone-induced oocyte maturation in Xenopus are not well understood. One of the first events is the activation of the MAPK cascade and the maturation-promoting factor (MPF). The latter triggers meiosis I resumption and meiosis II progression until the metaphase II arrest. The release of the metaphase II is mediated by the anaphase-promoting complex (APC)-dependent degradation of cyclin B. This degradation activity requires the APC activator Cdc20 that activates ubiquitination reactions by recruiting substrates to the APC. However, recent reports in different organisms involve other APC regulators during different phases of the meiotic cycle. Therefore, we investigated the role of another APC regulator, XCdh1 during the G2/M transition in meiosis I in the Xenopus oocyte. Here, we report that XCdh1 protein is expressed in oocytes. Besides, injection of specific XCdh1 antisense inhibits progesterone-induced G2/M transition that can be rescued by adding back the purified human Cdh1 protein. On the other hand, ectopic expression of low levels of XCdh1 protein has a positive effect on the G2/M transition by facilitating this process. Moreover, the sole injection of XCdh1 mRNA triggers Mos protein synthesis and biphosphorylation of MAPK in absence of progesterone. Altogether, these data show that XCdh1 has a positive role during the G2/M transition in the oocyte. According to our results, its role could be independent of the APC.     

The complete primary structure of the boar spermadhesin AQN-1, a carbohydrate-binding protein involved in fertilization.

Gamete recognition and adhesion are essential steps in the complex process of fertilization. In mammals and in other species, increasing evidence indicates that carbohydrate-binding proteins on the sperm surface play a pivotal role as counter-receptors for certain oligosaccharide moieties attached to the oocyte zona pellucida glycoproteins. Although different sperm-associated zona-pellucida-binding proteins have been identified in a number of species, few of them have been isolated and structurally characterized. In this paper we report the primary structural characterization of AQN-1, a 12-kDa boar-sperm-associated carbohydrate-binding and zona-pellucida-binding protein. The molecular mass of AQN-1 was determined by time-of-flight plasma-desorption mass spectrometry. Determination of its amino acid sequence and location of disulphide bridges were accomplished by a combination of proteochemical and mass spectrometric methods. The primary structure of AQN-1 failed to show any significant similarity to the protein structures deposited with the Martinsried Institute for Protein Sequences data bank, indicating that it may belong to a novel protein family involved in fertilization. AQN-1 shares extensive structural, as well as functional, similarity with two other boar sperm zona-pellucida-binding proteins, AQN-3 and AWN, which we have recently characterized. To name this protein family, we have coined the term spermadhesin. Our data may be relevant for identification of spermadhesins in other species, and thus may contribute to a better understanding of the species-specific sperm-egg recognition mechanism.     

Protein phosphorylation and oocyte maturation : I. Induction of starfish oocyte maturation by intracellular microinjection of a phosphatase inhibitor, α-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. α-Naphthylphosphate (α-NP), a widely used phosphatase inhibitor/substrate, was found to induce oocyte maturation when microinjected intracellularly (50% maturation at 3.5 mM; 100% above 6 mM, final intracellular concentration) into oocytes of Marthasterias and Asterias but not of Astropecten. As 1-MeAde, α-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 α-NP-induced maturation (35–45 min) is, however, longer than the kinetics of 1-MeAde-induced maturation (18–20 min). The addition of α-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 α-NP, only α-NP was found capable of inducing maturation when microinjected into oocytes. α-NP triggers the appearance of MPF activity in the cytoplasm of oocytes into which it has been injected. Although α-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 α-NP-induced maturation is inhibited by simultaneous microinjection of protein phosphatase-2A; also, α-NP, classically used as an inhibitor of acid and alkaline phosphatases, is able to inhibit protein phosphatases 1 and 2 A. The addition of α-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.     

Steroids involved with final oocyte maturation in the winter flounder.

A number of androgens and progestogens including 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20-P) were examined in female winter flounder as possible maturation inducing steroids (MIS). During final oocyte maturation serum levels of testosterone (T) and 17 beta-hydroxy-5 beta-androsten-3-one (5 beta-T) peaking at over 200 ng/ml and pregnenolone (PE) at 40 ng/ml were the predominant steroids found from each major group. High levels of T and 5 beta-T were correlated with oocyte stages characterized by germinal vesicle migration. Of the PEs measured, maximum serum levels of PE, 3 beta,17 alpha-hydroxy-5-pregnen-20-one (17-PE) and 3 beta,17 alpha, 20 beta-dihydroxy-5-pregnene (17,20-PE) were found during later oocytes stages associated with germinal vesicle breakdown. Levels of 17,20-P, an established MIS in most fish, were almost non-detectable (less than 0.1 ng/ml serum) in females throughout all stages of final oocyte maturation. Incubations of ovarian follicles in vitro with physiological concentrations of T and 5 beta-T indicated that these steroids could induce all stages of final oocyte maturation. Similar in vitro incubations showed that 17-PE and 17,20-PE were only effective on germinal vesicle breakdown. The principal conclusions are that T, 5 beta-T and the PEs can be considered as MISs in winter flounder and the PE pathway predominates during the final stages of oocyte maturation in winter flounder in contrast to progesterones which predominate in other fish species, mostly salmonids, studies to date.     

Purification and structural determination of an inhibitor of starfish oocyte maturation from a Bacillus species.

Inhibitors of bacterial origins of starfish oocyte maturation were sought to obtain biologically active substances which act on either hormonal signal transduction or cell cycle regulation. An oocyte maturation-inhibiting substance found in culture fluid of a Bacillus species was purified to homogeneity. This substance possessed the nature of a detergent and specifically inhibited 1-methyladenine-induced oocyte maturation (50% inhibitory concentration, 3.3 microM) but not dithiothreitol-induced maturation. Its total structure was established to be the lactone of 3-hydroxy-13-methyltetradecanoyl-Glu-Leu-Leu-Val-Asp-Leu -Leu through COOH of the carboxy-terminal Leu. This structure is identical to surfactin, although although the configuration of the substance's amino acid residues has not yet been determined. Surfactin was shown to be identical with this substance in its inhibitory effect on starfish oocyte maturation as well as its chromatographic and electrophoretic properties. Therefore, it was concluded that the oocyte maturation-inhibiting substance produced by a Bacillus species is surfactin.     

Scapinin,a putative protein phosphatase-1 regulatory subunit associated with the nuclear nonchromatin structure

It is thought that the nuclear nonchromatin structures, such as the nuclear matrix and lamina, play regulatory roles in gene expression. In this study, we identified an insoluble protein that was associated with the chromatin-depleted nuclear structure of proliferating human leukemia HL-60 cells. Preparation of the chromatin-depleted nuclear structure, referred to as the nuclear matrix-intermediate filament scaffold (Fey, E., Krochmalnic, G., and Penman, S. (1986) J. Cell. Biol. 102, 1654-1665), involved cell extraction using a series of buffers containing Triton X-100, DNase I, and 2 M NaCl. A yeast two-hybrid assay revealed that this protein bound to the catalytic subunit of protein phosphatase-1 (PP1). Furthermore, it inhibited PP1 activity in vitro. We therefore named it scapinin (scaffold-associated PP1 inhibiting protein). cDNA cloning revealed that scapinin had two splicing variants of 448 amino acids (scapinin-S) and 518 amino acids (scapinin-L). Scapinin was down-regulated by differentiation in HL-60 cells. These results suggest that scapinin is a putative regulatory subunit of PP1 and is involved in transformed or immature phenotypes of HL-60 cells. We also describe the presence of scapinin family proteins from worm to human.     

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.     

The X-ray structure of the FMN-binding protein AtHal3 provides the structural basis for the activity of a regulatory subunit involved in signal transduction

BACKGROUND: The Arabidopsis thaliana HAL3 gene product encodes for an FMN-binding protein (AtHal3) that is related to plant growth and salt and osmotic tolerance. AtHal3 shows sequence homology to ScHal3, a regulatory subunit of the Saccharomyces cerevisae serine/threonine phosphatase PPz1. It has been proposed that AtHal3 and ScHal3 have similar roles in cellular physiology, as Arabidopsis transgenic plants that overexpress AtHal3 and yeast cells that overexpress ScHal3 display similar phenotypes of improved salt tolerance. The enzymatic activity of AtHal3 has not been investigated. However, the AtHal3 sequence is homologous to that of EpiD, a flavoprotein from Staphylococcus epidermidis that recognizes a peptidic substrate and subsequently catalyzes the alpha, beta-dehydrogenation of its C-terminal cysteine residue. RESULTS: The X-ray structure of AtHal3 at 2 A resolution reveals that the biological unit is a trimer. Each protomer adopts an alpha/beta Rossmann fold consisting of a six-stranded parallel beta sheet flanked by two layers of alpha helices. The FMN-binding site of AtHal3 contains all the structural requirements of the flavoenzymes that catalyze dehydrogenation reactions. Comparison of the amino acid sequences of AtHal3, ScHal3 and EpiD reveals that a significant number of residues involved in trimer formation, the active site, and FMN binding are conserved. This observation suggests that ScHal3 and EpiD might also be trimers, having a similar structure and function to AtHal3. CONCLUSIONS: Structural comparisons of AtHal3 with other FMN-binding proteins show that AtHal3 defines a new subgroup of this protein family that is involved in signal transduction. Analysis of the structure of AtHal3 indicates that this protein is designed to interact with another cellular component and to subsequently catalyze the alpha,beta-dehydrogenation of a peptidyl cysteine. Structural data from AtHal3, together with physiological and biochemical information from ScHal3 and EpiD, allow us to propose a model for the recognition and regulation of AtHal3/ScHal3 cellular partners.     

Rearrangement of the actin-spectrin cytoskeleton during oocyte maturation of the starfish Asterias amurensis

Actin and spectrin localization in the oocytes of the starfish Asterias amurensis at hormonal induction of maturation until the destruction of the germinal vesicular membrane has been investigated by immunocytochemical and immunoblotting methods. In immature oocytes, spectrinlike protein and actin are detected to be colocalized in the undermembranous area of the cytoplasm and nuclear membrane. 1-Methyladenine causes redistribution of these proteins into intracellular structures. The actin-spectrin cytoskeleton rearrangement is shown to start at the animal pole of the oocyte and to spread then to its vegetative pole.     

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.     

Cyclin synthesis, modification and destruction during meiotic maturation of the starfish oocyte

The pattern of protein synthesis in oocytes of starfish Marthasterias glacialis changes during 1-methyladenine-induced meiotic maturation. One of the newly synthesized proteins, a major 54-kDa polypeptide, was synthesized continuously after activation but was destroyed abruptly just before appearance of the polar bodies at each meiotic division. This protein thus resembles the cyclin proteins identified in cleaving sea urchin and clam embryos. RNA extracted from oocytes before and after maturation encoded virtually identical polypeptides when translated in the reticulocyte lysate. However, there was poor correspondence between the in vitro translation products and the labelling pattern of intact cells. There was no exact in vitro counterpart to the in vivo-labelled cyclin. Instead, a major polypeptide of 52 kDa was seen which appears to be a precursor of the 54-kDa form of cyclin. The 52-kDa polypeptide was identified as cyclin by hybrid arrest of translation. Cyclin mRNA is ot translated to a significant extent before oocyte activation and is present in oocytes as nonadenylated form. It becomes polyadenylated when the oocytes mature. This behavior is also seen in the case of the mRNA for the small subunit of ribonucleotide reductase, another abundant maternal mRNA whose translation is activated at maturation.