Dissociation of protamine-DNA complexes by Xenopus nucleoplasmin and minichromosome assembly in vitro

Nucleoplasmin, an acidic thermostable protein abundant in the nucleus of Xenopus laevis oocytes, has been found to dissociate complexes of pUC19 DNA and protein phi 1, an intermediate protamine present in ripe sperm from the mollusc Mytilus edulis. Cruder preparations of nucleoplasmin, such as the amphibian oocyte S150 extract and its thermostable fraction, also dissociate the heterologous DNA-phi 1 complexes and, in addition, promote the assembly of plasmid DNA into a minichromosome displaying regular nucleosomal periodicity, as revealed by micrococcal nuclease digestion. In contrast, purified nucleoplasmin complemented with rat hepatocyte core histone octamers in the presence of DNA topoisomerase I, although capable of inducing nucleoprotein formation onto the complexed DNA, fails to position nucleosomes at the native spacings seen in chromatin in vivo. These data favour the existence of a general mechanism to bring about, in a concerted manner, removal of sperm-specific nuclear proteins and reconstitution of somatic chromatin following fertilization.     

Essential role for gamma-tubulin in the acentriolar female meiotic spindle of Drosophila

Microtubule nucleation in vivo requires gamma-tubulin, a highly conserved component of microtubule-organizing centers. In Drosophila melanogaster there are two gamma-tubulin genes, gammaTUB23C and gammaTUB37C. Here we report the cytological and molecular characterization of the 37C isoform. By Western blotting, this protein can only be detected in ovaries and embryos. Antibodies against this isoform predominantly label the centrosomes in embryos from early cleavage divisions until cycle 15, but fail to reveal any particular localization of gamma-tubulin in the developing egg chambers. The loss of function of this gene results in female sterility and has no effect on viability or male fertility. Early stages of oogenesis are unaffected by mutations in this gene, as judged both by morphological criteria and by localization of reporter genes, but the female meiotic spindle is extremely disrupted. Nuclear proliferation within the eggs laid by mutant females is also impaired. We conclude that the expression of the 37C gamma-tubulin isoform of D. melanogaster is under strict developmental regulation and that the organization of the female meiotic spindle requires gamma-tubulin.     

Histone H1 reduces the frequency of initiation in Xenopus egg extract by limiting the assembly of prereplication complexes on sperm chromatin

Somatic histone H1 reduces both the rate and extent of DNA replication in Xenopus egg extract. We show here that H1 inhibits replication directly by reducing the number of replication forks, but not the rate of fork progression, in Xenopus sperm nuclei. Density substitution experiments demonstrate that those forks that are active in H1 nuclei elongate to form large tracts of fully replicated DNA, indicating that inhibition is due to a reduction in the frequency of initiation and not the rate or extent of elongation. The observation that H1 dramatically reduces the number of replication foci in sperm nuclei supports this view. The establishment of replication competent DNA in egg extract requires the assembly of prereplication complexes (pre-RCs) on sperm chromatin. H1 reduces binding of the pre-RC proteins, XOrc2, XCdc6, and XMcm3, to chromatin. Replication competence can be restored in these nuclei, however, only under conditions that promote the loss of H1 from chromatin and licensing of the DNA. Thus, H1 inhibits replication in egg extract by preventing the assembly of pre-RCs on sperm chromatin, thereby reducing the frequency of initiation. These data raise the interesting possibility that H1 plays a role in regulating replication origin use during Xenopus development.     

Decondensation of sperm nuclei in vitro

The urine of five patients with three distinct diseases ("I Cell disease" and two new types of mucolipidosis) contains sialic acid-rich oligosaccharides in a high amount: 50- to 500-fold the normal. The structure of the major components are as follows: alphaAcNeu(2 leads to 6)betaGal(1 leads to 4)betaGlcNac(1 leads to 2)alphaMan(1 leads to 3)betaMan(1 leads to 4)GlcNac,[alphaAcNeu(2 leads to 6)]betaGal(1 leads to 4)betaGlcNAc(1 leads to 2)alphaMan(1 leads to 3)[betaGal(1 leads to 4)betaGlcNac(1 leads to 2)alphaMan(1 leads to 6)]betaMan(1 leads to 4)GlcNAc and alphaAcNeu(2 leads to 6)betaGal(1 leads to 4)betaGlcNAc(1 leads to 2)alphaMan(1 leads to 3)[alphaAcNeu(2 leads to 6)betaGal(1 leads to 4)betaGlcNAc(1 leads to 2)alphaMan(1 leads to 6)]betaMan(1 leads to 4)GlcNAc. These results suggest that a deficit in alpha-neuraminidase is associated to these three different disorders and that an endo-beta-D-N-acetylglucosaminidase is able to release sialyoligosaccharides by splitting the sialylglycans of glycoproteins.     

Neural crest formation in Xenopus laevis: mechanisms of Xslug induction

A study of the induction of the prospective neural crest in Xenopus laevis embryos has been carried out, using the expression of Xslug as a specific marker for the neural crest. We have analyzed the competence and the specification of the neural crest. The competence to express Xslug was analyzed using two different approaches: (1) in vitro culture of conjugates of dorsal mesoderm and ectoderm taken from embryos at different ages and (2) grafts of equivalent pieces of ectoderm in the neural fold region of a gastrula or a neurula. Similar results were obtained with both methods: the ectoderm loses the competence to respond to neural fold induction at the end of gastrulation. Neural crest specification was analyzed by culturing a region of the ectoderm that contained the prospective neural crest and analyzing Xslug expression. Our results show that neural folds are specified autonomously to express Xslug by the end of gastrulation. By grafting labeled neural plate into lateral epidermis we have shown that neural crest can be induced by an interaction between neural plate and epidermis. Furthermore, neural crest cells come from both tissues. We have discarded the possibility that these neural crest cells are induced by a signal coming from the underlying lateral plate, by a homeogenetic signal coming from the host neural plate, or by regeneration of crest cells from the dissected neural plate. We propose a model to explain how the neural crest cells are induced at the border of the neural plate in X. laevis.     

Xenopus laevis fertilisation: analysis of sperm motility in egg jelly using video light microscopy

OBJECTIVE: To evaluate the origin of pseudomyxoma peritonei (PMP) in Chinese women. METHODS: The clinicopathologic features of 15 cases of PMP were reviewed. Immunostaining using antibodies for CK7 and CK20 was performed in the ovarian, appendiceal and peritoneal lesions of these cases. RESULTS: Appendiceal pathology was documented in five cases, including four mucinous cystadenoma and one simple mucocele. Eight ovarian tumors were found, including seven mucinous cystadenocarcinomas of low malignant potential and one mucinous cystadenoma. Synchronous ovarian and appendiceal lesions were discovered in three cases. One patient had adenocarcinoma of the pancreas. The origin of mucin production was not known in four cases with metastatic adenocarcinoma found in two of them. Immunoreactivity for CK20 was demonstrated in the tissues derived from the peritoneum, ovary, appendix and pancreas while only 23% (3 out of 13 women) of the peritoneal lesions and 33% (2 out of 6 women) of the ovarian tumors were immunoreactive for CK7. CONCLUSIONS: PMP is a heterogeneous lesion, which may develop from mucinous metaplasia of the peritoneum or from appendiceal, or ovarian lesions. Careful examination of the ovary and appendix with performance of appendectomy is advised in every case of PMP. Immunohistochemical examination of the peritoneal, ovarian or appendiceal lesions using antibodies, in particular that for CK7 would help in defining the origin of mucin production.     

Male immunologic infertility: sperm performance on in vitro fertilization

OBJECTIVE: To analyze sperm performance in a group of patients with male immunologic infertility treated with IVF-ET. DESIGN: Retrospective clinical study. SETTING: Patients attending a private IVF clinic. PATIENT(S): The study group comprised seven men with significant levels of surface-bound antisperm antibodies treated in nine IVF cycles. The control group comprised nine couples with female tubal infertility and no indication of male factor infertility treated on the same cycle. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Fertilization rate, early embryonic development, implantation, and clinical pregnancy rate (PR). RESULT(S): Forty-six (44.2%) of 104 inseminated oocytes were fertilized in the study group compared with 65 (84.4%) of 77 in the control group, which was a significant difference. Surface-bound antisperm antibodies significantly inhibited early embryonic cleavage in the study group (13 [28.3%] of 46 embryos with at least 3 blastomeres) compared with the control group (41 [63.1%] of 65 embryos, with at least 3 blastomeres). The percentage of good-quality embryos (grades 1 and 2) was similar in the study and control groups (71.7% and 78.5%, respectively). The percentage of poor-quality embryos (grade 4 and two pronuclei) was higher in the study group compared with the control group (13.9% versus 9.2%, respectively); however, the difference was not significant. The implantation rate and clinical PR were lower in the study group (3% and 11%, respectively) compared with the control group (9.5% and 44%, respectively), but the difference was not statistically significant. CONCLUSION(S): The fertilization rate and early embryonic cleavage of human embryos was found to be reduced significantly in patients with high levels of surface-bound antisperm antibodies. Moreover, embryonic quality and the PR may be compromised by the presence of significant levels of surface-bound antisperm antibodies.     

Complete replication of human sperm genome in egg extracts from Xenopus laevis

Significant amounts of D-aspartate (Asp) are found in mammalian tissues and D-Asp is presumed to play some significant, but as yet undefined physiological role. However, it is not known whether D-Asp is synthesized in mammals. In this study, we addressed this issue in cultured rat pinealocytes, parenchymal cells of the pineal gland, which contain significant amounts of D-Asp. Biosynthesis of D-Asp was found to be minimal to non-existent in cultured rat pinealocytes. We then investigated the mechanism of uptake of D-Asp into these cells and its consequent effect on cell function. D-Asp was efficiently taken up into cells, in a time- and dose-dependent manner. Interestingly, the L-Asp levels in the cells and media decreased concomitantly with the uptake of D-Asp. This decrease was not due to D-Asp cytotoxicity, since the cellular levels of othernted. D-Serine and D-alanine were not taken up efficiently into the cells and the cellular levels of L-serine and L-alanine were unchanged. Also, immunocytochemical staining with anti-D-Asp antibody showed that D-Asp, which had been taken up into the cells, was dispersed throughout the cytoplasm. In response to norepinephrine stimulation, pinealocytes, which had been pretreated with D-Asp released D-Asp as well as L-Asp. In these cells, norepinephrine-induced secretion of melatonin, a pineal hormone, was suppressed. The mechanism of this suppression is discussed here.     

Spliceosome assembly: the unwinding role of DEAD-box proteins

Splicing factors belonging to the DEAD-box superfamily can affect RNA conformation in vitro. These proteins may control the dynamic changes in RNA base-pairing interactions during spliceosome assembly.     

Hailey-Hailey disease keratinocytes: normal assembly of cell-cell junctions in vitro

The blisters in the inherited disorder, Hailey-Hailey disease, may be caused by defective epidermal junctional complexes. We evaluated these structural complexes in vivo and in vitro. We induced a vesicular lesion in the apparently normal skin of a patient with Hailey-Hailey disease and studied a biopsy of this lesion by transmission electron microscopy. To determine whether acantholysis was related to a defect in the number or assembly of intercellular junctions, we cultured Hailey-Hailey disease keratinocytes in medium containing 0.1 mM Ca2+ and increased the [Ca2+] to 1.1 mM in order to induce assembly of cell-cell junctions. Keratinocytes were examined by double immunofluorescence with antibodies to the desmosome protein, desmoplakin, and the adherens junction protein, vinculin, at intervals after the increase in [Ca2+]. Characteristic Hailey-Hailey disease histopathology was observed by electron microscopy of the patient's skin after trauma, but we found no splitting of desmosomes. Based on the location, intensity, and rate of change of immunofluorescent staining, Hailey-Hailey and normal keratinocytes did not differ in their ability to assemble desmosomes and adherens junctions. Furthermore, we observed no significant morphologic differences between normal and Hailey-Hailey keratinocytes cultured in low and high [Ca2+]-containing media; Hailey-Hailey cells contained abundant normal-appearing desmosomes in 1.1 mM [Ca2+]. Since Hailey-Hailey disease keratinocytes can assemble normal-appearing adherens junctions and desmosomes in vitro, the functional defect may not lie in assembly of cell-cell adhering junctions, or additional perturbation may be required to expose the defect.     

Functional expression of sperm angiotensin II type I receptor in Xenopus oocyte: modulation of a sperm Ca2+-activated K+ channel

gamma-Hydroxybutyric acid (GHB) is an abused substance that occurs naturally in the basal ganglia. Electrophysiological recordings of membrane voltage and current were made to characterize the effects of GHB on dopamine neurons in the ventral tegmental area of the rat midbrain slice. Perfusate containing GHB caused a concentration-dependent membrane hyperpolarization (EC50 = 0.88 +/- 0.21 mM) and a reduction in input resistance (EC50 = 0.74 +/- 0.21 mM). The highest concentration of GHB studied (10 mM) hyperpolarized neurons by 20 +/- 3 mV and reduced input resistance by 58% +/- 9%. Changes in membrane potential and input resistance were blocked by the gamma-aminobutyric acid antagonist CGP-35348 (300 microM), but neither bicuculline (30 microM) nor strychnine (10 microM) was an effective antagonist. Voltage-clamp recordings demonstrated that GHB (1 mM) evoked 80 +/- 6 pA of outward current (at -60 mV) that reversed at -110 mV (in 2.5 mM K+). Increasing concentrations of extracellular K+ progressively shifted the reversal to more depolarized potentials. In tetrodotoxin (0.3 microM) and tetraethylammonium (10 mM), depolarizing voltage steps (to -30 mV) evoked calcium-dependent current spikes that were completely blocked by GHB (1 mM). These data suggest that GHB is an agonist at gamma-aminobutyric acid receptors and would be expected to inhibit DA release by causing K+-dependent membrane hyperpolarization.     

Glucocorticoid receptor inhibits microtubule assembly in vitro

The effect of glucocorticoid hormones, purified glucocorticoid receptor (GR) and purified heat shock protein M(r) 90,000 (hsp90) on microtubule (MT) assembly in vitro was tested by a spectrophotometric MT assembly assay and electron microscopy. GR significantly prolonged the nucleation phase, slowed down the assembly rate and reduced the maximal amplitude of MT assembly compared with control. The effects were partially reversed by the addition of glucocorticoid hormone. GR associated with MTs. These results indicate that GR affects MT assembly in vitro, which may be a functional correlate to the structural association of GR with MTs. This implies that factors affecting GR may affect MT assembly in vivo.     

Inability to detect Chlamyodomonas microtubule assembly in vitro: possible implications to the in vivo regulation of microtubule assembly

It has been demonstrated that the in vitro assembly of microtubules from Chlamydomonas preparations does not occur under a wide range of conditions, including those efficacious for mammalian brain tubulin. This incompetence of Chlamydomonas extracts to form microtubules is independent of the tubulin concentration, the presence of added nucleotides or an added seed, temperature, or the concentration of divalent cation. However, an amorphous aggregate was observed under certain conditions, who composition was mainly tubulin. The in vitro reassembly of microtubules in gerbil brain extracts is inhibited by Chlamydomonas preparations. Fractionation of the Chlamydomonas extracts by column chromatography suggests that the inhibitory component is Chlamydomonas tubulin itself. The mechanism of this inhibition is unknown, but reassembly experiments indicate that the 2 types of tubulins cannot copolymerize. We suggest that the Chlamydomonas tubulin, derived from a cytoplasmic pool, requires to be activated prior to its in vivo polymerization into microtubules.     

Sperm aster in rabbit zygotes: its structure and function

Experimental neurosis was achieved in fifteen rabbits (both fixed on a stand and unrestrained) by sudden, drastic enhancement of a pain electrocutaneous stimulation used as reinforcement, at the stage of a stable elaborated avoidance reflex. The experimental neurosis was characterized by a prolonged (one to two months) disturbance of conditioned activity, greater motor restlessness, predominance of the fear type reactions, the animals' reduced weight, higher pulse and respiratory rate. Dynamics of the respiratory rate and brain bioelectrical activity became chaotic. Simultaneously, bioelectrical activity revealed greater similarity of the processes in the cortex and subcortical structures at one and the same moment. Analysis of the electrogram frequency spectra of different brain formations, pointed to an enhancement in all frequency ranges (with the exception of gamma), with a clear predominance of the 6 to 10 c/s band which was most distinctly expressed in the amygdaloid structures.     

XCTK2: a kinesin-related protein that promotes mitotic spindle assembly in Xenopus laevis egg extracts

We used a peptide antibody to a conserved sequence in the motor domain of kinesins to screen a Xenopus ovary cDNA expression library. Among the clones isolated were two that encoded a protein we named XCTK2 for Xenopus COOH-terminal kinesin 2. XCTK2 contains an NH2-terminal globular domain, a central alpha-helical stalk, and a COOH-terminal motor domain. XCTK2 is similar to CTKs in other organisms and is most homologous to CHO2. Antibodies raised against XCTK2 recognize a 75-kD protein in Xenopus egg extracts that cosediments with microtubules. In Xenopus tissue culture cells, the anti-XCTK2 antibodies stain mitotic spindles as well as a subset of interphase nuclei. To probe the function of XCTK2, we have used an in vitro assay for spindle assembly in Xenopus egg extracts. Addition of antibodies to cytostatic factor-arrested extracts causes a 70% reduction in the percentage of bipolar spindles formed. XCTK2 is not required for maintenance of bipolar spindles, as antibody addition to preformed spindles has no effect. To further evaluate the function of XCTK2, we expressed XCTK2 in insect Sf-9 cells using the baculovirus expression system. When purified (recombinant XCTK2 is added to Xenopus egg extracts at a fivefold excess over endogenous levels) there is a stimulation in both the rate and extent of bipolar spindle formation. XCTK2 exists in a large complex in extracts and can be coimmunoprecipitated with two other proteins from extracts. XCTK2 likely plays an important role in the establishment and structural integrity of mitotic spindles.     

Xenopus laevis sperm receptor gp69/64 glycoprotein is a homolog of the mammalian sperm receptor ZP2

Little is known about sperm-binding proteins in the egg envelope of nonmammalian vertebrate species. We report here the molecular cloning and characterization of a recently identified sperm receptor (gp69/64) in the Xenopus laevis egg vitelline envelope. Our data indicate that the gp69 and gp64 glycoproteins are two glycoforms of the receptor and have the same number of N-linked oligosaccharide chains but differ in the extent of O-glycosylation. The amino acid sequence of the receptor is closely related to that of the mouse zona pellucida protein ZP2. Most of the sequence conservation, including a ZP domain, a potential furin cleavage site, and a putative transmembrane domain are located in the C-terminal half of the receptor. Proteolytic cleavage of the gp69/64 protein by a cortical granule protease during fertilization removes 27 amino acid residues from the N terminus of gp69/64 and results in loss of sperm binding to the activated eggs. Similarly, we find that treatment of eggs with type I collagenase removes 31 residues from the N terminus of gp69/64 and has the same effect on sperm binding. The isolated and purified N terminus-truncated receptor protein is inactive as an inhibitor of sperm-egg binding. Earlier studies on the effect of Pronase digestion on receptor activity suggest that this N-terminal peptide may contain an O-linked glycan that is involved in the binding process. Based on these results and the findings on the primary structure of the receptor, a pathway for the maturation and secretion of gp69/64, as well as its inactivation following fertilization, is proposed.     

A role for Xenopus Frizzled 8 in dorsal development

The cGMP-binding cGMP-specific phosphodiesterase (PDE-5) contains distinct catalytic and allosteric binding sites, and each is cGMP-specific. Cyclic nucleotide phosphodiesterase inhibitors, such as 3-isobutyl-1-methylxanthine (IBMX), are believed to compete with cyclic nucleotides at the catalytic sites of these enzymes, but the portion of PDE-5 that accounts for interaction of either of these inhibitors of the substrates themselves with the catalytic domain of the enzymes has not been identified. IBMX was derivatized to yield the photoaffinity probe 8([3-125I,-4-azido]-benzyl)-IBMX, which is referred to as 8(125IAB)-IBMX. This probe was incubated with partially purified recombinant bovine PDE-5. After UV irradiation and SDS-PAGE, a single radiolabeled band that coincided with the position of PDE-5 was visualized on the gel, and the photoaffinity labeling of PDE-5 was linear with increasing concentration of the 8(125IAB)-IBMX. Prominent Coomassie blue-stained bands other than PDE-5 were not labeled significantly. The photoaffinity labeling was progressively blocked by cGMP at concentrations higher than 10 microM, whereas cAMP or 5'-GMP exhibited only weak inhibitory effects. Other compounds that are believed to interact with the PDE-5 catalytic site, including IBMX, cIMP, and beta-phenyl-1,N2-etheno-cGMP (PET-cGMP), also inhibited the photoaffinity labeling in a concentration-dependent manner. The IC50 of PET-cGMP for inhibition of photoaffinity labeling was 10 microM, which compared favorably with an IC50 of 5 microM for inhibition of PDE-5 catalytic activity by this compound. It is concluded that the interaction of this photoaffinity probe with PDE-5 is highly specific for the catalytic site over the allosteric binding sites of PDE-5 and could prove useful in studies to map the catalytic site of PDE-5.     

Determinants of weddellite formation: chondroitin sulfates and citrate determine weddellite formation in vitro

In synthetic urine (SU), addition of oxalate tends to form monohydrates of calcium oxalate. However, addition of oxalate to natural urine preferably forms calcium oxalate dihydrate (COD). Urine apparently contains a determinant for COD formation. To identify the determinant, the effects of pH, temperature, oxalate, calcium, urate, citrate, magnesium, sulfate and chondroitin sulfates (CS) on calcium oxalate crystal formation were studied. Lower temperatures, higher oxalate concentrations and higher pH favored COD formation in a SU. Mixed CS in the presence of citrate were the most decisive determinant of COD formation. Substitution of CS for agar and gelatin produced similar results, indicating that the colloidal effect of the macromolecules determines COD formation. Identification of the determinants led to a simple, reproducible method of COD formation in SU without natural urine. Addition of strontium to SU resulted in dodecahedral bipyramids. Interpenetration twinning of bipyramids occur within seconds of the crystal formation.     

Conditions for fibronectin fibril formation in the early Xenopus embryo

A longitudinal study on exposure to tobacco smoke among adolescents was carried out in Turin (North-Western Italy) in January-February 1992 and in January-February 1993. In 1992, 394 schoolchildren aged 14-16 years were enrolled in a study protocol which consisted in answering a standardized questionnaire, measurement of urinary cotinine and testing of lung function (flow-volume curve--[FVC] and forced expiratory volume in I sec.--[FEV1]). In 1993, 333 schoolchildren from the same group repeated the survey. By comparison to urinary cotinine, findings obtained showed a reduction of increase, from 1992 to 1993, of -0.57% (p = 0.082) for FVC, and -0.66% (p = 0.05) for FEV1. Assuming that the systematic selection bias did not seem to have occurred, findings, obtained from a multiple regression analysis, showed that active and passive exposure to tobacco smoke, as measured by urinary cotinine, had a significant effect on lung growth (as measured by FEV1) in adolescents; this effect, though small, was dose-related.