Stability of cyclin B protein during meiotic maturation and the first mitotic cell division in mouse oocytes

This report examines in detail the metabolism of the cyclin protein B1 during meiotic maturation and following the activation of mature mouse oocytes using immunoprecipitation of the radiolabelled protein. The net synthesis of cyclin B increases progressively during meiotic maturation, reaching its maximum levels at least 1 h before oocytes exit into metaphase of meiosis II (MII). This increase correlates with the rise in cdc2 kinase activity reported previously and suggests an association between the length of the first meiotic M phase (MI) and the net synthesis of cyclin B, that seems to regulate the time required for the cdc2 kinase to reach its maximum activity. Moreover, no marked degradation of cyclin B was observed before the MI to MII transition and that which occurs does so independently of the presence of microtubules, which are essential for cyclin degradation during metaphase II arrest and exit of oocytes into interphase of the first mitotic cell cycle. Cyclin B is degraded rapidly during the transitions MI to MII, MII to the first mitotic interphase and MII to an abortive third metaphase state (MIII). However, whilst its degradation was incomplete during the MI to MII transition, virtually no cyclin B protein was detected following both the MII to interphase and MII to MIII transitions. Thus, the decision of oocytes to exit into MIII, or interphase is not controlled at the level of cyclin B degradation. Lastly, in aging, non-activated oocytes, the net synthesis of cyclin B declines. Whereas, in activated eggs cultured in parallel although the rate of net synthesis declines initially, it is effectively ‘rescued’ being two-fold greater than in non-activated oocytes of an equivalent age. This gradual fall in the net synthesis of cyclin B observed in aging oocytes may contribute to the increasing ease with which they become activated, compared to recently ovulated oocytes.     

P-loop Flexibility in Na+ Channel Pores Revealed by Single- and Double-cysteine Replacements

Replacement of individual P-loop residues with cysteines in rat skeletal muscle Na⁺ channels (SkM1) caused an increased sensitivity to current blockade by Cd²⁺ thus allowing detection of residues lining the pore. Simultaneous replacement of two residues in distinct P-loops created channels with enhanced and reduced sensitivity to Cd²⁺ block relative to the individual single mutants, suggesting coordinated Cd²⁺ binding and cross-linking by the inserted sulfhydryl pairs. Double-mutant channels with reduced sensitivity to Cd²⁺ block showed enhanced sensitivity after the application of sulfhydryl reducing agents. These results allow identification of residue pairs capable of approaching one another to within less than 3.5 Å. We often observed that multiple consecutive adjacent residues in one P-loop could coordinately bind Cd²⁺ with a single residue in another P-loop. These results suggest that, on the time-scale of Cd²⁺ binding to mutant Na⁺ channels, P-loops show a high degree of flexibility.     

Diel spawning periodicity of red snapper Lutjanus campechanus in the northern Gulf of Mexico

Ovaries of red snapper Lutjanus campechanus were examined histologically to determine rates of oocyte maturation, diel spawning periodicity and whether lunar cycle influenced spawning rhythm. Hydration of red snapper oocytes began during the mid‐morning hours; c . 5 h was necessary for oocytes to become fully hydrated and ovulation occurred no more than 5 h after oocytes attained full hydration. Appearance of fresh postovulatory follicles after 1330 hours and the absence of hydrated oocytes after 1830 hours signified that red snapper spawning occurred during this 5 h period. In addition, evidence of a peak in spawning was seen near 1600 hours. Postovulatory follicles degenerated within a 24 h time period. A lunar spawning cycle was not evident.     

Identification and localization of α- and β-spectrins in oocytes of three Antarctic teleosts: Trematomus bernacchii, Trematomus newnesi (Nototheniidae) and Chionodraco hamatus (Channichthyidae)

The presence and localization of α- and β-spectrins and of the spectrin cross-linking protein actin were investigated, in previtellogenic oocytes of three species of Antarctic teleosts: the two red-blooded nototheniids, Trematomus bernacchii and Trematomus newnesi , and the channichthyid, the ice fish Chionodraco hamatus . Analyses by western blotting indicated that these species had an unusual abundance of spectrin isoforms and that they were characterized by rather low molecular masses. The immunocytochemistry in situ demonstrated that α- and β-spectrins showed a variable pattern of localization that clearly depended on both the species considered and the stage of oocyte differentiation. In particular, the two Trematomus spp. showed a distribution of spectrins absolutely comparable and rather different from that of C. hamatus . The evidences collected confirmed that channichthyids have isolated early from the group of red-blooded species and suggest that in notothenioids significant changes might have occurred in spectrin genes and in their protein products.     

Functional Changes in Rat Nigral GABAA Receptors Induced by Degeneration of the Striatonigral GABAergic Pathway: An Electrophysiological Study of Receptors Incorporated into Xenopus Oocytes

Abstract: Expression of rat brain γ-aminobutyric acid type A (GABA_A) receptors in Xenopus laevis oocytes can be achieved by injection of the oocytes with synaptosomes. This approach has now been applied to evaluate changes in the function of nigral GABA_A receptors after degeneration of the striatonigral GABAergic pathway induced by the unilateral infusion of kainic acid into the rat striatum. Ten days after striatal injection, synaptosomal membranes were prepared from the substantia nigra and introduced into oocytes. Nigral GABA_A receptors incorporated into the oocyte cell membrane were then characterized electrophysiologically under voltage-clamp conditions. The maximal amplitude of GABA-induced Cl^? currents in oocytes injected with synaptosomes from denervated substantia nigra was twice that observed in oocytes injected with synaptosomes from control substantia nigra. The concentration of GABA required for the half-maximal response did not differ between the two groups of oocytes. In addition, the potentiation of GABA-induced currents by the benzodiazepine diazepam (1 µM) and the steroid derivative allopregnanolone (3 µM) was increased by ～65 and 60%, respectively, in oocytes injected with synaptosomes from denervated substantia nigra compared with those injected with control synaptosomes. The concentrations of diazepam and allopregnanolone giving half-maximal responses were not affected by denervation. In contrast, the inhibitory effects of the benzodiazepine receptor inverse agonists FG 7142 (10 µM) and 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylic acid ethyl ester (1 µM) were reduced by 48 and 38%, respectively, after denervation. These results indicate that the up-regulation of nigral GABA_A receptors induced by degeneration of the striatonigral GABAergic pathway is associated with an increased efficacy of positive allosteric modulators, such as benzodiazepines and steroids, and with a reduced efficacy of negative allosteric modulators such as β-carbolines.     

The ultrastructure of the nuclear envelope of amphibian oocytes

Summary In order to investigate the chemical composition of the nuclear pore complexes isolated nuclei from matureXenopus laevis oocytes were manually fractioned into nucleoplasmic aggregates and the nuclear envelopes. The whole isolation procedure takes no more than 60–90 sec, and the pore complexes of the isolated envelopes are well preserved as demonstrated by electron microscopy. Minor nucleoplasmic and cytoplasmic contaminations associated with the isolated nuclear envelopes were determined with electron microscopic morphometry and were found to be quantitatively negligible as far as their mass and nucleic acid content is concerned. The RNA content of the fractions was determined by direct phosphorus analysis after differential alkaline hydrolysis. Approximately 9% of the total nuclear RNA of the matureXenopus egg was found to be attached to the nuclear envelope. The nonmembranous elements of one pore complex contain 0.41×10^–16 g RNA. This value agrees well with the content estimated from morphometric data. The RNA package density in the pore complexes (270×10^–15 g/³) is compared with the nucleolar, nucleoplasmic and cytoplasmic RNA concentration and is discussed in context with the importance of the pore complexes for the nucleo-cytoplasmic transport of RNA-containing macromolecules.Additionally, the results of the chemical analyses as well as of the³H-actinomycin D autoradiography and of the nucleoprotein staining method of Bernhard (1969) speak against the occurence of considerable amounts of DNA in the nuclear pore complex structures.The author thanks Miss Ulrika Lempert, Miss Marianne Winter, and Miss Sigrid Krien for skilful technical help as well as Dr. W. W. Franke for many helpful discussions. The work has been supported by a Deutsche Forschungsgemeinschaft grant given to Dr. W. W. Franke (SFB Molgrudent, 46).     

Cryopreservation of Unfertilized Mouse Oocytes: The Effect of Replacing Sodium with Choline in the Freezing Medium

Although embryo cryopreservation has become commonplace in many species, effective methods are not available for routine freezing of unfertilized eggs. Cryopreservation-induced damage may be caused by the high concentration of sodium ions in conventional freezing media. This study investigates the effect of a newly developed low-sodium choline-based medium (CJ2) on the ability of unfertilized, metaphase II mouse eggs to survive cryopreservation and develop to the blastocyst stagein vitro.Specifically, the effects of cooling to subzero temperatures, thawing rate, LN₂plunge temperature, and equilibration with a low-sodium medium prior to freezing are examined. In contrast to cooling to 23, 0, or −7.0°C in a sodium-based freezing medium (ETFM), cooling in CJ2 had no significant negative effect on oocyte survival or development. Oocytes frozen in CJ2 survived plunging into LN₂from −10, −20, or −33°C at significantly higher rates than oocytes frozen in ETFM. With the protocol used (1.5 M PrOH, 0.1 M sucrose, −0.3 C/min, plunging at −33°C) rapid thawing by direct submersion in 30°C water was more detrimental to oocyte survival than holding in air for 30 or 120 s prior to transfer to water. Equilibration of unfertilized oocytes with a low-sodium medium prior to cryopreservation in CJ2 significantly increased survival and blastocyst development. These results demonstrate that the high concentration of sodium in conventional freezing media is detrimental to oocyte cryopreservation and show that choline is a promising replacement. Reducing the sodium content of the freezing medium to a very low level or eliminating sodium altogether may allow oocytes and other cells to be frozen more effectively.     

Peculiarities of the antioxidant and radioprotective effects of hydrated C60 fullerene nanostuctures in vitro and in vivo

Aqueous solutions of highly stable supramolecular donor–acceptor complexes of chemically nonmodified pristine C₆₀ fullerene molecules with H₂O molecules (hydrated C₆₀ fullerene–C₆₀HyFn) and their labile nano-sized clusters were examined for their antioxidant effects on removal of hydroxyl radicals (OH) and protecting DNA against oxidative damage induced by ionizing radiation in vitro. The suppressing influence of C₆₀HyFn on the formation of OH-radicals in water exposed to X-rays at doses of 1–7 Gy was assessed by determination of oxidation levels of coumarin-3-carboxylic acid. C₆₀HyFn demonstrates apparent antiradical activity in vitro in the range of concentrations of 10⁻¹¹–10⁻⁶ M. Paradoxically, the OH-removing efficacy of C₆₀HyFn was in reverse correlation with fullerene concentration. It was hypothesized that the antiradical action of C₆₀HyFn in water medium generally is due to a “nonstoichiometric” mechanism, supposedly to a hydrated free radical recombination (self-neutralization), which is catalyzed by specific water structures ordered by C₆₀HyFn. With the use of 8-oxoguanine as a marker of oxidative damage to DNA, it has been demonstrated that C₆₀HyFn in concentrations of 10⁻⁷–10⁻⁶ M protects nucleic acids against radical-induced damage. The second part of the present study was aimed to evaluate the overall radioprotective efficacy of C₆₀HyFn in doses of 0.1 or 1 mg/kg b.w. injected intraperitoneally to mice either 1 h before or 15 min after lethal dose exposure of the X-ray (7 Gy) irradiation. Survival rate of the mice was observed at 30 day intervals after irradiation, while the weight gains of experimental animals were monitored as well. The most significant protective effect was demonstrated when 1 mg/kg dosage of C₆₀HyFn was administered before irradiation. The outcome of the substance testing is 15% survival rate of irradiated animals at 30 days of observation, and prevention of noticeable weight loss characteristic for radiation impact, versus unprotected control animals. In conclusion, results of the study obviate that the apparent protective action of C₆₀HyFn in vivo is determined by its considerable ability to decrease X-ray-generated reactive oxygen species. Based on the results and that neat C₆₀ is nontoxic, actually in the hydrated form, without side effects and with sufficient radioprotective effects in low doses, C₆₀HyFn may be considered as a novel antioxidant agent, which substantially diminishes the harmful effects of ionizing radiation.     

Identification and characterization of a novel amphioxus dopamine D1-like receptor

Dopamine receptors function to control many aspects of motor control and other forms of behaviour in both vertebrates and invertebrates. They can be divided into two main groups (D₁ and D₂) based on sequence similarity, ligand affinity and effector coupling. However, little is known about the pharmacology and functionality of dopamine receptors in the deuterostomian invertebrates, such as the cephalochordate amphioxus ( Branchiostoma floridae) which has recently been placed as the most basal of all the chordates. A bioinformatic study shows that amphioxus has at least three dopamine D₁-like receptor sequences. One of these receptors, AmphiD₁/β, was found to have high levels of sequence similarity to both vertebrate D₁ receptors and to β-adrenergic receptors. Here, we report on the cloning of AmphiD₁/β from an adult amphioxus cDNA library, and its pharmacological characterization subsequent to its expression in both mammalian cell lines and Xenopus oocytes. It was found that AmphiD₁/β has a similar pharmacology to vertebrate D₁ receptors, including responding to benzodiazepine ligands. The pharmacology of the receptor exhibits 'agonist-specific coupling' depending upon the second messenger pathway to which it is linked. Moreover, no pharmacological characteristics were observed to suggest that AmphiD₁/β may be an amphioxus orthologue of vertebrate β-adrenergic receptors.