Hox genes in the echiuroid Urechis unicinctus

An echiuroid species, Urechis unicinctus, was surveyed for Hox genes using polymerase chain reaction with homeobox-specific degenerate primers. We identified nine distinct homeodomain-containing gene fragments. These nine fragments were classified by comparative analysis. This analysis revealed that this echiuroid possessed at least three Hox genes from the anterior group, five from the central group, and one from the posterior group.Sung-Jin and Dae-Hee Lee contributed equally to this work.An erratum to this article can be found at     

The localization of the nuclear protein pool in Xenopus oocytes

It has been demonstrated previously that nuclear proteins in Xenopus oocytes are synthesized in the cytoplasm and maintained in a cellular pool. The present study was performed to determine if any portion of this pool is associated specifically with the nuclear envelope. This was accomplished by first micro-injecting oocytes with [³H]leucine; at various times after injection, nuclear envelope and nucleoplasmic fractions were run on SDS-polyacrylamide gels. In this way labeled polypeptides available in the envelope fraction could be compared to polypeptides which were subsequently incorporated into the nucleoplasm. No evidence was obtained that the nuclear protein pool is associated with the envelope.     

The role of the maturation-promoting factor in controlling protein synthesis in Xenopus oocytes

Oocyte cytosol, containing maturation-promoting factor activity, induces a twofold increase in the rate of protein synthesis as well as inducing germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes. In the current study, it is shown that the cytosol activity responsible for inducing the increase in protein synthesis can be separated from the activity that induces GVBD in recipient oocytes.     

Nuclear protein synthesis in animal and vegetal hemispheres of Xenopus oocytes

Experiments were conducted to determine if nuclear proteins are preferentially synthesized in the vicinity of the nucleus, a factor which could facilitate nucleocytoplasmic exchange. Using Xenopus oocytes, animal and vegetal hemispheres were separated by bisecting the cells in paraffin oil. It was initially established that protein synthesis is not affected by the bisecting procedure. To determine if nuclear protein synthesis is restricted to the animal hemisphere (which contains the nucleus), vegetal halves and enucleated animal halves were injected with [3H]leucine and incubated in oil for 90 min. The labeled cell halves were then fused with unlabeled, nucleated animal hemispheres that had been previously injected with puromycin in amounts sufficient to prevent further protein synthesis. Thus, labeled polypeptides which subsequently entered the nuclei were synthesized before fusion. Three hours after fusion, the nuclei were isolated, run on two-dimensional gels, and fluorographed. Approximately 200 labeled nuclear polypeptides were compared, and only 2 were synthesized in significantly different amounts in the animal and vegetal hemispheres. The results indicate that nuclear protein synthesis is not restricted to the cytoplasm adjacent to the nucleus.     

Major protein changes during vitellogenesis and maturation of Fundulus oocytes

The protein content of various size follicles was measured in Fundulus heteroclitus and indicated four phases of increase relative to follicle volume: Phases I (previtellogenic; estimated to be less than 0.01 mg/mm3), II (vitellogenic; 0.20 mg/mm3), III (early maturation; 0.03 mg/mm3), and IV (late maturation; 0 mg/mm3). A pronounced and rapid size increase occurs during maturation due to hydration, but protein uptake, which was also documented cytologically, contributes to about 16% of the volume increment during early maturation. Protein incorporation appears to stop abruptly at the time of germinal vesicle breakdown, most likely reflecting an altered physiological state of the oocyte. SDS-polacrylamide gel electrophoretic patterns of various size follicles indicated that five major protein bands (molecular weights = 122, 103, 45, 26, and 20 k) accumulate during vitellogenesis and presumably are proteolytically derived from a 200-kDa vitellogenin precursor. During maturation, the 122- and 45-kDa proteins disappear and several new, lower molecular weight bands appear. Proteolysis of specific yolk proteins may thus help generate part of the osmotic pressure gradient required for water uptake during oocyte maturation.     

Intracellular transport of vitellogenin in Xenopus oocytes: An autoradiographic study

The role of primordial yolk platelets (PYPs) in the transport of the yolk precursor vitellogenin to the yolk platelets in Xenopus laevis oocytes has been demonstrated by electron microscopic autoradiography. Within 20 min after exposure of the oocyte to ³H-labeled-vitellogenin, silver grains are associated with small PYPs which are formed by the fusion of endosomes. At 40 min after incorporation of ³H-labeled vitellogenin, autoradiographic silver grains are associated with larger PYPs and with the superficial layer of yolk platelets. Thus, the results demonstrate that PYPs are an intermediate in the transport of vitellogenin from endosomes to yolk platelets. These observations are consonant with the general hypothesis that vitellogenin first associates (binds?) with the plasma membrane, then is incorporated by endocytosis into endosomes which fuse to form PYPs, and finally the contents of the PYPs are eventually deposited into yolk platelets.     

Turnover of endogenous, microinjected, and sequestered protein in Xenopus oocytes

Pulse-labeled oocyte proteins were found to have a maximum average half-life of 73 h. In general, larger peptides underwent degradation at a faster rate than smaller peptides. In this respect, oocytes are similar to most other cells. Microinjected ¹²⁵I-labeled bovine serum albumin (BSA) was degraded over a 40 h period with a half-life of 20–30 h, regardless of the method of protein labeling, culture medium employed, size of oocyte microinjected, or hormonal history of the oocyte. The last two results, if applicable to oocyte proteins in general, imply that protein catabolism is constant throughout the later stages of oogenesis and that growth is primarily regulated by a stimulation of anabolism. Individual proteins microinjected into oocytes undergo rates of degradation consistent with turnover rates obtained in other systems. Sequestered ¹²⁵I-labeled BSA is only partially (40%) degraded, which indicates that, unlike microinjected ¹²⁵I-labeled BSA, it has access to a cytoplasmic compartment (yolk platelets?) within which it is relatively stable.     

Chromosomal interchange in mature oocytes of drosophila

Detachments of a reversed acrocentric compound-X chromosome (C(I)RA) resulting from interchange with a doubly-marked Y chromosome (y⁺Yy⁺) were induced by irradiating mature, stage-14 oocytes in 5-day-old, adult virgin females of Drosophila melanogaster. The period of egg laying after irradiation was limited to 12 h to prevent inclusion in the sample of eggs that were irradiated in immature stages. It was confirmed that some rearrangements must be completed prior to the completion of division I, since chromosomes that regularly assort apart at division I—C(I)RA and y⁺Yy⁺—have been able to interchange parts to permit the recovery of half-translocations. Further evidence is afforded by the higher recovery of proximal (capped) that of distal (captured) X's, from which can be inferred nonrandom segregation of heteromorphic dyads.The absence of fractionation effects in mature oocytes has been accounted for in previous publications by postulating delayed repair, but the delay cannot extend beyond the initiation of chromosome movement in division I of meiosis. Our data, however, do not exclude the possibility that some interchanges might be completed postmeootically, but neither do they suggest it. It is argued that the kinds of data that have been derived from mature Drosophila oocytes are not yet adequate to form a basis for choosing between breakage-first and exchange models of interchange.The significance of these findings in relation to the kinds of aberrations that can be recovered from irradiated mature oocytes is briefly discussed.     

An investigation into the mutagenic after-effect of butadiene diepoxide usingNeurospora crassa

Summary Butadiene diepoxide (DEB) was used to induce reverse mutations in anad-3A mutant ofNeurospora crassa. It was found that the mutagenic action of DEB continued when the twice washed and resuspended conidia were left standing in water at room temperature. The after-effect can be prevented if the conidia are kept stirred by shaking or gaseous bubbling for 4–5 hours. The after-effect does not develop if the conidia are plated immediately after washing, or if they are kept at 0–4°C until plating. The experimental data indicate that the after-effect is caused by traces of DEB, or by mutagenic reaction products of DEB, which are not readily removed by the ordinary washing procedure, but which may be removed by diffusion from the cells when these are kept suspended for a prolonged period.With 3 Figures in the Text     

An amber mutation in the gene rpsA for ribosomal protein S1 in Escherichia coli

Summary An amber mutation has been induced in the gene rpsA (which codes fo ribosomal protein S1) of Escherichia coli K-12 strain in the presence of an amber suppressor (supD) and mutations sueA, sueB and sueC that additively enhance the efficiency of suppression. That the amber mutation has occurred in the gene rpsA was confirmed by complementation with a plasmid which carried the wild-type allele of rpsA. The mutation is lethal in the absence of an amber suppressor, indicating that ribosomal protein S1 is indispensable to E. coli.     

Mitochondrial DNA synthesis in oocytes of Xenopus laevis

Mitochondria isolated from stage 3 (about half-grown) oocytes of Xenopus laevis exhibit a DNA synthetic rate in vitro of 2.35 ± 0.28 pg/oocyte/h. Similarly, stage 6 (full-grown) oocyte mitochondria synthesize DNA (mtDNA) at 0.28 ± 0.02 pg/oocyte/h. By comparison, the rate of mtDNA synthesis by intact stage 6 oocytes following microinjection of [³H]-dTTP was calculated to be 0.43 ± 0.08 pg/oocyte/h, indicating that the observed in vitro rates may represent minimum values. Measurements of DNA polymerase activity associated with mitochondria isolated from stage 3 oocytes are almost three times those recorded with stage 6 oocyte mitochondria. It appears that active replication of complete mtDNA molecules, which accompanies accumulation of mitochondria by the egg, is terminated midway through oogenesis.     

Analysis of zeins' ER retention in Xenopus oocytes

Zeins, maize storage proteins, are retained in the endoplasmic reticulum (ER) during the intracellular protein targeting process. Hydrophobic interaction has been postulated as the driving force of zeins' aggregation and retention in the ER. Recently, a class of zein (the 27K zein) has been proposed to facilitate zeins' ER retention by anchoring to the ER membrane. This study investigated the significance of the two proposed mechanisms toward zeins' ER retention using Xenopus oocyte. Following injection of the total or 27K zein mRNA, zein's movement within the ER was analyzed based upon the extent of diffusion to the non-injected oocyte half. This study indicates that the total zeins freely move within the lumen of the ER, thus, suggesting that the intermolecular aggregation, leading to insolubility and exclusion from the ER lumenal fluid, may not be essential for zeins' ER retention. This study also suggests that the 27K zein may not facilitate zeins' ER retention by virtue of an anchor to the ER membrane based on its free movement in the ER. Free movement of the total and 27K zeins, under conditions where zein aggregates should form, necessitates a reevaluation of the mechanisms responsible for zein polypeptides' ER retention and protein body formation.     

Non-spindle microtubule organizing centers in metaphase II-arrested mouse oocytes

A human autoantiserum (5051) directed against pericentriolar material (PCM) was used to study the distribution of microtubule-organizing centers (MTOCs) in the oocyte and during the first cell cycle of mouse development. In oocytes, the PCM was found not only at the poles of the barrel-shaped metaphase II spindle but also at many discrete loci around the cytoplasm near the cell cortex. The spindle poles were also composed of several PCM foci. In metaphase-arrested eggs only the PCM foci located near the chromosomes acted as MTOCs. However, after reduction of the critical concentration for tubulin polymerization by taxol, the cytoplasmic PCM foci were also found to be associated with nucleation of microtubules. After fertilization the cortical PCM foci remained in a peripheral position until the end of the S phase, when they appeared to migrate centrally towards the pronuclei. At prometaphase of the first mitotic division, numerous MTOCs were found around the two sets of chromosomes; these MTOCs then aligned to form two bands on either side of the metaphase plate of the first mitosis.     

Ultrastructure of human mature oocytes after vitrification

Since the introduction of human assisted reproduction, oocyte cryopreservation has been regarded as an attractive option to capitalize the reproductive potential of surplus oocytes and preserve female fertility. However, for two decades the endeavor to store oocytes has been limited by the not yet optimized methodologies, with the consequence of poor clinical outcome or of uncertain reproducibility. Vitrification has been developed as the promising technology of cryopreservation even if slow freezing remains a suitable choice. Nevertheless, the insufficiency of clinical and correlated multidisciplinary data is still stirring controversy on the impact of this technique on oocyte integrity. Morphological studies may actually provide a great insight in this debate. Phase contrast microscopy and other light microscopy techniques, including cytochemistry, provided substantial morpho-functional data on cryopreserved oocyte, but are unable to unraveling fine structural changes. The ultrastructural damage is one of the most adverse events associated with cryopreservation, as an effect of cryo-protectant toxicity, ice crystal formation and osmotic stress. Surprisingly, transmission electron microsco py has attracted only limited attention in the field of cryopreservation. In this review, the subcellular structure of human mature oocytes following vitrification is discussed at the light of most relevant ultrastructural studies.Key words: oocyte, MII, vitrification, ultrastructure, TEM, human.     

Hormone-induced parthenogenetic activation of mature starfish oocytes

1-Methyladenine, which has been previously shown to be the hormone responsible for meiosis reinitiation in starfish oocytes, triggers parthenogenetic activation when applied to matured starfish oocytes after emission of the second polar body and formation of the pronucleus. In Marthasterias glacialis and Asterias rubens oocytes parthenogenetic activation includes elevation of a fertilization membrane, cleavage and the formation of normal bipinnaria larvae. Activation is likely to result from 1-methyladenine interaction with the category of stereospecific membrane receptors involved in meiosis reinitiation, since structural requirements of this compound are identical for both biological responses. Appearance of oocyte responsiveness to 1-MeAde after, but not before emission of the second polar body cannot be accounted for by their increased sensitivity to intracellular Ca²⁺ at that time, although it is shown that Ca²⁺ mediates hormone effect in inducing parthenogenetic activation. Pretreatment of immature oocytes with the free hormone in excess strongly inhibits the 1-methyladenine-induced parthenogenetic activation of the oocytes when they have completed maturation.It is suggested that reappearance of 1-MeAde sensitivity when oocytes form a pronucleus depends either upon recruitment or new receptor units or on the reactivation of pre-existing inactivated receptors at this stage of oocyte maturation.     

Evidence for steroid metabolism during the in vitro induction of maturation in oocytes of Rana pipiens

Oocytes of Rana pipiens exposed to exogenous progesterone in order to induce maturation have been observed to extensively metabolize this hormone. When progesterone was injected directly into the oocytes, they did not mature, but similar metabolism of progesterone occurred. The metabolites have been tentatively identified as the 5α-reduced derivatives, 5α-pregnanedione, 5α-pregnan-20α-ol-3-one, and 5α-pregnan-3β, 20α-diol, and the pathway of conversion has been examined. Samples of these steroids obtained from commercial sources and those extracted from progesterone-treated oocytes were effective in inducing maturation when added to the medium. Evidence is presented which suggests that steroid metabolism is not a prerequisite for maturation and that the metabolites like progesterone must interact with the oocyte surface to be effective.