Induction of prematurely condensed chromosomes from testicular cells of the mouse

Mitotic CHO cells and mouse testicular cells were fused with polyethylene glycol. Several types of prematurely condensed chromosomes were observed. From chromosome morphology it was possible to determine that most of the PCC represented mouse cells. Labeling of either the CHO cells in vitro or the testicular cells in vivo with ³H-TdR prior to fusion also demonstrated that the PCC were derived from the mouse cells. In some PCC, 20 chromosomes could be counted, the haploid number for mouse. It is assumed that these PCC were induced in mouse spermatid nuclei.     

Induction of chromosome damage by Neurospora endonuclease in repair-inhibited quiescent normal human fibroblasts

The purpose of these experiments was to determine the role of double-strand breaks in chromosome aberration formations. Quiescent normal human fibroblasts were treated with 3 μM nitrogen mustard and then allowed to repair their DNA damage for 24 h prior to cell fusion and induction of premature chromosome condensation. The extent of chromosome damage was determined in the G1 prematurely condensed chromosomes (G1 PCC). The presence of cytosine arabinoside and hydroxyurea during the repair period in order to accumulate single-strand DNA breaks resulted in an increase in the chromosome-break frequency. Treatment of these repair-inhibited cells with single-strand-specific neurospora endonuclease during fusion to change single-strand lesions into double-strand breajs resulted in a doubling of the aberration frequency. These results support the notion that double-strand breaks are important in chromosome-aberration formation.     

Chromosomes of caryophyllidean cestodes: Diploidy, triploidy, and parthenogenesis in Glaridacris catostomi

Mitotic and meiotic chromosomes were studied in Giemsa post-stained Feulgen squashes of testicular, ovarian, and vitelline cells. G. catostomi is 2n = 20 with eight pairs of metacentrics and two pairs of submetacentrics in its karyotype. The fundamental number of chromosome arms is FN = 40. At mitotic metaphase the mean total chromosome length (TCL) of the complement was 111·4 μm; the largest chromosomes were 8 μm (7% TCL) and the smallest 3 μm (3% TCL). Karyotype variation was not found among five diploid populations from New York State and Canada. Meiotic activity was abundant in spermatogenesis which proceeds as usual with sperm developing after two meiotic divisions. Chiasma frequency at diakinesis was 37/cell; as many as five chiasmata were observed in a single bivalent. A triploid ‘race’ (3n = 30) of G. catostomi was discovered in Bozenkill Creek near Albany, New York. Karyotype analysis confirms the triploid nature of these variants. Spermatogenesis in triploids is abnormal to the point of complete failure. The presence of an extra set of chromosomes has resulted in a breakdown of meiosis with subsequent sterility of the male system. Eggs remain unfertilized and parthenogenetic reproduction is presumed to occur. The possible origins of this unusual condition and its evolutionary implications are discussed. Seventeen figures, two tables of measurements, and a system of chromosome nomenclature supplement the paper.     

Chromatin differentiation between Theobroma cacao L. and T. grandiflorum Schum

A comparative analysis of mitotic chromosomes of Theobroma cacao (cacao) and T. grandiflorum (cupuaçu) was performed aiming to identify cytological differences between the two most important species of this genus. Both species have symmetric karyotypes, with 2n = 20 metacentric chromosomes ranging in size from 2.00 to 1.19 μm (cacao) and from 2.21 to 1.15 μm (cupuaçu). The interphase nuclei of both species were of the arreticulate type, displaying up to 20 chromocentres, which were more regularly shaped in cacao than in cupuaçu. Prophase chromosomes of both species were more condensed in the proximal region, sometimes including the whole short arm. Both species exhibited only one pair of terminal heterochromatic bands, positively stained with chromomycin A ₃ , which co-localized with the single 45S rDNA site. Each karyotype displayed a single 5S rDNA site in the proximal region of another chromosome pair. Heterochromatic bands were also observed on the centromeric/pericentromeric regions of all 20 chromosomes of cacao after C-banding followed by Giemsa or DAPI staining, whereas in cupuaçu they were never detected. These data suggest that the chromosomes of both species have been largely conserved and their pericentromeric chromatin is the only citologically differentiated region.     

Palm Chromosome Studies Facilitated by Pollen Culture on a Colchicine-Lactose Medium

This technique is very useful where pollen is readily available and when roots or microspore mother cells are difficult to obtain or to process. It provides a relatively uniform means of studying chromosomes in a great number of species. Pollen is collected from buds at anthesis or the day before and sown on a medium containing H₃BO₃, 100 ppm; colchicine, 0.04%; lactose, 12%; gelatin, 5%; egg albumen, 1 drop in 10 ml. The gametic chromosome complement is studied at mitosis of the generative cell in the pollen tube. In species which have sufficiently large chromosomes it is possible to construct idiograms for comparative studies. All palm species here reported have a haploid complement of n = 18, and the chromosomes range in length from 0.5-3.5 μ.     

Prematurely condensed human sperm chromosomes after in vitro fertilization (IVF)

Summary During an in vitro fertilization (IVF) program 122 inseminated eggs showing polar body extrusion, but neither formation of pronuclei nor cell cleavage were analysed cytogenetically. Nine of these eggs showed prematurely condensed sperm chromosomes of the G₁-phase (G₁-PCC) besides the haploid set of maternal metaphase II chromosomes. This phenomenon can be explained by the permanent arrest of the oocytes at metaphase II after sperm penetration and hence the continuing presence of cytoplasmic chromosome condensing factors which lead to the induction of PCC in the sperm nucleus. The overall frequency of this aberrant type of fertilization was calculated to be in the order of 3–4% of all in vitro fertilized eggs.     

Isolation of leishmanial parasites from a wild caught Anopheles gambiae mosquito in Kenya

A total of 232 mosquitoes were collected and dissected for leishmanial parasites in the Baringo District, Kenya. Anopheles gambiae sensu lato comprised 90.9% of the sample. One female A. gambiae was found to be infected with leishmanial promastigotes. The parasites when injected into Balb C mice caused skin lesions characterized by heavy amastigote infections. The average size of the parasite was: body length, 11.7 ± 0.19 μm; width, 1.3 ± 0.04 μm; flagellum length, 15.5 ± 0.28 μm.     

Length of human C-bands in relation to the degree of chromosome condensation

Summary The C-band length of human chromosome 1 in prophase and prematurely condensed interphase chromosomes is relatively shorter than in metaphase chromosomes. However, even in chromosomes with the same degree of contraction the absolute length of the C-band varies considerably. This allocyclic behaviour of human constitutive heterochromatin has to be kept in mind if C-bands of different individuals are compared.Sponsored by the Deutsche Forschungsgemeinschaft (Sp 144)     

Ultrastructure of a novel tube-forming, intracellular parasite of dinoflagellates: Parvilucifera prorocentri sp. nov. (Alveolata, Myzozoa)

We have characterized the intracellular development and ultrastructure of a novel parasite that infected the marine benthic dinoflagellate Prorocentrum fukuyoi. The parasite possessed a combination of features described for perkinsids and syndineans, and also possessed novel characters associated with its parasitic life cycle. Reniform zoospores, about 4 μm long, possessed a transverse flagellum, alveoli, a refractile body, a mitochondrion with tubular cristae, a syndinean-like nucleus with condensed chromatin, micronemes, bipartite trichocysts with square profiles (absent in perkinsids) and oblong microbodies. Like Parvilucifera, the zoospores also possessed a shorter posterior flagellum, a heteromorphic pair of central microtubules in the anterior axoneme and a reduced pseudoconoid positioned directly above an orthogonal pair of basal bodies. Early developmental stages consisted of a sporangium about 5–15 μm in diam that contained spherical bodies and amorphous spaces. The undifferentiated sporangium increased to about 20–25 μm in diam before being enveloped by a wall with a convoluted mid-layer. The sporangium differentiated into an unordered mass of zoospores that escaped from the cyst through a pronounced germ tube about 4–5 μm in diam and 10–15 μm long. Weakly developed germ tubes have been described in Perkinsus but are absent altogether in Parvilucifera and syndineans. Comparison of these data with other myzozoans led us to classify the parasite as Parvilucifera prorocentri sp. nov., Myzozoa. Although we were hesitant to erect a new genus name in the absence of molecular sequence data, our ultrastructural data strongly indicated that this parasite is most closely related to perkinsids and syndineans, and represents an intriguing candidate for the cellular identity of a major subclade of Group I alveolates.     

Taxonomic characterization of two marine peritrichous ciliates, Epicarchesium corlissi n. sp. and Pseudovorticella jiangi n. sp. (Ciliophora: Peritrichia), from northern China

Two new marine peritrich ciliates, Epicarchesium corlissi n. sp. and Pseudovorticella jiangi n. sp., were discovered in mariculture waters on the coast of northern China near Qingdao. Their morphology, infraciliature and silverline system were investigated based on both living and silver-impregnated specimens. E. corlissi is characterized as follows: marine Epicarchesium with dichotomously branched stalk; zooids elongate, approximately 60–70×25–35 μm in vivo; peristomial collar double-folded; macronucleus J-shaped; single, small contractile vacuole ventrally positioned; more than 60 striations between peristome and aboral trochal band, 13–18 from aboral trochal band to scopula; abstomal end of row 1 of infundibular polykinety 3 terminating at same level as rows 2 and 3 of infundibular polykinety 3; rows 2 and 3 of infundibular polykinety 3 much longer than row 1 and converging adstomally with infundibular polykinety 1. The new species P. jiangi is diagnosed as follows: marine Pseudovorticella; zooid inverted bell-shaped, approximately 80×60 μm in vivo and with a broad, flat, thin peristomial collar that measures approximately 90 μm across; pellicle with transparent cortical vesicles; macronucleus J-shaped; number of silverlines between peristome and aboral trochal band 20–24, from aboral trochal band to scopula 9–11; abstomal end of row 1 of infundibular polykinety 3 diverges from the other two rows of this polykinety and ends alongside row 3 of infundibular polykinety 2.     

Meiotic chromosome missegregation during apyrene meiosis in the gypsy moth,Lymantria dispar,is preceded by an aberrant prophase I

The gypsy moth, Lymantria dispar, produces two structurally and genetically distinct types of spermatozoa. The eupyrene spermatozoa are genetically haploid and structurally typical. The apyrene spermatozoa are anucleate and structurally different from eupyrene spermatozoa. To understand further the events contributing to meiotic chromosome missegregation in apyrene spermatocytes, we examined the progression of meiosis in these cells with respect to their eupyrene counterparts. Chromosomal bouquet formation and fusion of nucleolar organizing regions are disrupted in apyrene nuclei. In addition, the chromatin of apyrene nuclei is prematurely and extremely condensed compared with that of eupyrene nuclei. An antibody to the conserved synaptonemal complex protein 3 (SCP3) labeled eupyrene pachytene chromosomes, but not apyrene pachytene chromosomes. In addition, apyrene meiotic spindles are missing a subset of microtubules, which likely include kinetochore microtubules. Because the condensation behavior of meiotic chromatin in apyrene spermatocytes deviates from that of eupyrene spermatocytes, we examined the appearance and distribution of the phosphorylated form of histone H3, but no significant differences in histone H3 phosphorylation were found between apyrene and eupyrene spermatocytes. We argue that because a pachytene checkpoint is not initiated in apyrene spermatocytes, this system may provide a way to understand better the underlying biochemical connections between pairing, recombination, synapsis, kinetochore assembly and segregation of chromosomes during meiosis in a higher eukaryote.     

Structural and viral association comparisons of bovine zonae pellucidae from follicular oocytes, Day-7 embryos and Day-7 degenerated ova

Bovine zonae pellucidae (ZP) from follicular oocytes and from embryos and degenerated ova collected on Day 7 from superovulated cows were examined by scanning electron microscopy, by dimensional measurement, and by total protein determination. The number of plaque-forming units (PFU) of infectious bovine rhinotracheitis virus (IBRV) that were associated with ZP-intact embryos/ova from each of the 3 sources after in vitro exposure was also determined.

Scanning electron microscopy revealed that the surfaces of Day-7 embryos and degenerated ova were smoother than those of follicular oocytes. Mean dimensional measurements of the diameter/thickness of the ZP from follicular oocytes, Day-7 embryos, and degenerated Day-7 ova were 156.7 μm/12.3μm, 161.3μm/12.6μm, and 158.9μm/12.8μm, respectively. The mean total protein per ZP of follicular oocytes, embryos, and degenerated ova was 0.331 μg, 0.349 μg, and 0.254 μg, respectively. Considerable variability existed within groups, but significantly greater quantities of IBRV were associated with follicular oocytes (mean PFU/oocyte = 68.1) than with Day-7 embryos (mean PFU/embryo = 43.0; P<0.05) or with Day-7 ova (mean PFU/ovum = 31.9; P<0.01).

The reliability of using an assay for IBRV associated with nontransferable ova/embryos as an indicator of the presence or absence of the virus in transferable embryos from the same collection (Day 7) was supported. Although structural differences between the ZPs of follicular oocytes and Day-7 embryos were observed in this study, further investigation is needed to determine if there are differences in the protective function of the respective ZPs.     

Paraffin Section Thickness—A Direct Method of Measurement

Paraffin section thickness may be directly measured by re-embedding the sections wider consideration, cutting them again at right angles to the original plane of sectioning, and taking direct measurements with a filar micrometer after staining and mounting. Conditions and materials with which relatively un-distorted 3 and 5 μ sections were secured include (a) a hand-honed knife with a 23° facet bevel, set at a clearance angle of 7°, and (b) a hard paraffin (56-58°) embedding medium, preferably with 5% beeswax and 5% bayberry wax added. By taking direct measurements, it was found that bull testis tissue cut at a microtome setting of 10μ averaged 10.82 μ in thickness. Settings of 5 μ and 3 m resulted in sections averaging 5.25 and 3.31 μ in thickness respectively. Stages in sporogenesis of Onoclea sensibilis, Lewitsky fixed, were examined after sectioning at settings of 10, 5, and 3 μ to determine necessity for thin sections. For this material, it was indicated that mitochondrial preparations as thick as 10 μ were worthless, regardless of good fixation and proper staining. Three-micron sections give the best results.     

The presence of the potentially toxic genera Ostreopsis and Coolia (Dinophyceae) in the North Aegean Sea, Greece

The examination of macrophyte, water and sediment samples, collected at depths less than 1.5 m from 50 different sites along the North Aegean coasts, has revealed, for the first time in Greek coastal waters, the presence of two Ostreopsis species (O. ovata and O. cf. siamensis) and Coolia monotis in the majority of the sampling sites (94% and 100%, respectively). Other epiphytic dinoflagellates of the genera Prorocentrum and Amphidinium and diatoms were accompanying species in this epiphytic community. Morphometric features, plate formula and thecal ornamentation were used for species identification. O. ovata cells were smaller in dorsoventral (DV) diameter and width (W) (26.18–61.88 μm and 13.09–47.60 μm, respectively) in comparison with O. cf. siamensis (35.70–65.45 μm and 23.80–49.98 μm, respectively). In contrast, the anterioposterior (AP) diameter of O. cf. siamensis was smaller (14.28–26.18 μm) resulting in DV/AP ≈ 3, whereas the above ratio for O. ovata was less than 2 (AP ranging between 14.28–35.70 μm). Moreover, the theca of O. ovata cells was ornamented with scattered pores, which fluctuated in a wider range (0.07–0.32 μm) than those of O. cf. siamensis (0.23–0.29 μm). Coolia monotis cells were almost round with average DV diameter 26.88 μm, AP 25.66 μm and width 26.76 μm. Small and large cells were recorded in both field and culture populations of Ostreopsis spp. and C. monotis, while hyaline cysts were observed for O. ovata. The presence of O. ovata and O. cf. siamensis exhibited a clear seasonal pattern dominating (maximum abundance up to 4.05 × 10⁵ cells gr⁻¹ fwm) the period from midsummer to late autumn in years 2003 and 2004, while C. monotis was found also in winter and spring months.     

Galanin-induced relaxation in gastric smooth muscle cells is mediated by cyclic AMP

Galanin has numerous effects on gastrointestinal motility in different species; however, its cellular basis of action in mediating these effects is unclear. Dispersed gastric smooth muscle cells have been shown to possess high-affinity galanin receptors that increase cAMP and cause relaxation. Recent studies show some smooth muscle relaxants such as VIP cause relaxation by both cAMP-dependent and -independent mechanisms. It is unknown if galanin's cellular basis of relaxation is similar or different from that of VIP. To investigate galanin's relaxant effect and compare it to VIP's effect, dispersed smooth muscle cells from guinea pig stomach were prepared by collagenase digestion. The mean length in resting cells was 110 ± 2 μm and, with carbachol treatment, contracted to 89 ± 2 μm. VIP and galanin alone had no effect on cell length, but each caused a dose-dependent inhibition of carbachol-induced contraction and both had an EC₅₀ of 3–7 nM. Galanin (1 μM) and VIP (1 μM) increased cellular cAMP from 118 ± 10 pmol/10⁶ cells in control to 212 ± 14 and 214 ± 12 pmol/10⁶ cells, respectively. The protein kinase A inhibitor, Rp-cAMPS, at 100 μM, completely inhibited the relaxant effect of an EC₅₀ concentration of galanin (3 nM), but only inhibited that by VIP by 80% (p < 0.05). Adding the nitric oxide inhibitor, -NNA ( ), at 100 μM did not alter the length of resting cells or inhibit carbachol-induced contraction. However, -NNA (100 μM) decreased VIP-induced relaxation by 45%, whereas it had no effect on galanin-induced relaxation. To determine the ability of each peptide to activate nitric oxide, the incorporation of [³H]arginine into [³H]citrulline was determined. Galanin (1 μM) did not cause nitric oxide generation whereas VIP (1 μM) increased nitric oxide generation above the control by 97 ± 14% (p < 0.01). These results demonstrated that with galanin, in contrast to VIP, nitric oxide is not involved in its ability to cause gastric smooth muscle cell relaxation. The relaxant action of galanin can be accounted for completely by its ability to activate protein kinase A and therefore resembles recent results with β-adrenergic agents.     

Microspike-mediated particle transport towards the cell body during early spreading of 3T3 cells

Freshly trypsinized 3T3 cells send out microspikes of 0.2 μm diameter and up to 10 μm length within 20 min after attachment to a glass substratum. The microspikes move actively and eventually attach to the substratum. Subsequently, lamellae flow out between lines of attached microspikes. If, however, colloidal gold particles of 0.2–0.4 μm diameter and clusters of gold particles up to 4 μm in diameter are placed on the substratum and a microspike attaches to them, we observed two reactions of the microspikes to this contact. They either retract upon contact, transporting the attached particles to the cell surface at a speed of 0.2 μm/sec, or the particles flow toward the cell body while the microspike stays in place. This action results in the clearing of a circular area around each spreading cell before lamellae flow out. “Clearing” proceeds at serum concentrations between 1 and 20% and in concentrations of colchicine up to 20 μm/ml. In concentrations of cytochalasin B higher than 5 μg/ml, however, particle removal is completely inhibited, although the microspikes are still produced by the cell. Transmission electron microscopy shows that the microspikes contain mostly longitudinally oriented microfilaments and only a few microtubules, if any.     

Chromosome fission or duplication in Macrostomum lignano (Macrostomorpha, Plathelminthes) – remarks on chromosome numbers in 'archoophoran turbellarians'

Mitotic metaphase plates of a new species of the genus Macrostomum were studied using conventional Giemsa staining, Orcein staining and semi-thin sections stained after Richardson. Because of the ease in culturing it, this species has the potential to become the new model organism for developmental and evolutionary studies among the lower metazoa. The chromosome number was found to be 2 n  = 8. Reaching a relative length of 45.9% of the haploid genome size, the chromosomes of one pair were significantly larger than all other chromosomes. Of the smaller pairs, chromosomes of one pair were slightly larger (relative length of 21.3%) than the chromosomes of the remaining two pairs (each with a relative length of 16.4%). All chromosomes were metacentric (2 m  + 2 m  + 2 m  + 2 m ). For the first time, a diploid chromosome set of four pairs was described for the genus, as compared with previous studies showing predominantly 2 n  = 6 for 20 different Macrostomum species.