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MutS homolog 4 localization to meiotic chromosomes is required for chromosome pairing during meiosis in male and female mice.

Msh4 (MutS homolog 4) is a member of the mammalian mismatch repair gene family whose members are involved in postreplicative DNA mismatch repair as well as in the control of meiotic recombination. In this report we show that MSH4 has an essential role in the control of male and female meiosis. We demonstrate that MSH4 is present in the nuclei of spermatocytes early in prophase I and that it forms discrete foci along meiotic chromosomes during the zygotene and pachytene stages of meiosis. Disruption of the Msh4 gene in mice results in male and female sterility due to meiotic failure. Although meiosis is initiated in Msh4 mutant male and female mice, as indicated by the chromosomal localization of RAD51 and COR1 during leptonema/zygonema, the chromosomes fail to undergo normal pairing. Our results show that MSH4 localization on chromosomes during the early stages of meiosis is essential for normal chromosome synapsis in prophase I and that it acts in the same pathway as MSH5.

Animals↗

KL/KIT co-expression in mouse fetal oocytes.

The tyrosine kinase receptor, KIT, and its ligand, KL are important regulators of germ cell development. The aim of this study was to examine in detail the expression of the genes encoding these proteins (White and Steel, respectively) during the fetal period (14.5-18.5 days post coitum, dpc) and the two weeks after birth in mouse ovaries using the highly sensitive in situ reverse-transcriptase polymerase chain reaction (in situ RT-PCR). KL and KIT mRNAs were not detected in 14.5-15.5 dpc ovaries but, between 16.5 and 17.5 dpc, most of the oocytes in the outer regions of the ovaries positively stained for both mRNAs. The majority of the co-expressing oocytes were identified at the zygotene/pachytene stage of meiotic prophase I. At 18.5 dpc, positive staining for KL mRNA was present only in the somatic cells in the outer regions of the ovaries. At birth, faint KL mRNA-labelled somatic cells were mainly found in the central region of the ovaries and, by P7-14, a higher level of expression was detected in the follicle cells of one- and two-layered growing follicles. Between 17.5 dpc and birth, most of the oocytes expressed KIT mRNA and, from P7 onward, there was a considerable accumulation of transcripts in the growing oocytes. The results of in situ RT-PCR were confirmed by RT-PCR on purified populations of oocytes, and at protein level by means of immunohistochemistry. The co-expression of KL and KIT in a fraction of fetal oocytes suggests that the KL/KIT system, besides the well known paracrine functions on germ cells, may exert a novel autocrine role during the mid-stage of the oocyte meiotic prophase. The possibility that this autocrine loop plays a role in sustaining the survival of fetal oocytes in this stage is supported by the finding that the addition to the culture medium of anti-KL or anti-KIT antibodies led to a significant increase in oocyte apoptosis in the absence of exogenous KL.

Animals↗

[Hemodynamic collapse during off-pump coronary artery bypass grafting and optimization].

The present study addressed the incidence of unpredicted hemodynamic collapse in off-pump coronary artery bypass grafting (OPCAB). Since 1999, OPCAB was attempted in 114 patients without preoperative hemodynamic collapse to date. 95% patients of OPCAB were completed without percutaneous cardiopulmonary support (PCPS) and all patients were discharged. In this study, patients are divided into a prophase of 60 patients and an anaphase of 50 patients, in consideration of learning curve. As for the trouble in procedures, 12 patients of ventricular fibrillation, 6 patients of conversion to PCPS, 19 patients of bradycardia which were evaded in temporary pacing were observed. Patients with conversion to PCPS decreased in 5 patients (8%) in prophase and 1 patient (1.8%) in anaphase. Avoidance of hemodynamic collapse were prevention of ischemia by positive use of coronary perfusion, prophylactic intra-aortic balloon pumping (IABP) use for decrease of blood pressure, apical evacuation heart positioner for visual field security.

Adult↗

[Effects of accumulated temperature and planting density on tillering dynamics of forage rye].

Using equal design method, a field experiment was carried out at Wuqiao field station of China Agricultural University in Hebei Province to study the effects of different accumulated temperature before winter and planting density on tillering dynamics of forage rye (Secale cereale L.). The results showed that the tiller number per plant was affected by sowing date and seed density. In the prophase of forage rye, the tiller number per plant was mainly influenced by sowing date; while in the anaphase, it was mainly determined by seed density. Generally, earlier and fewer sowing norm could raise the tiller number in individual plant. In the prophase, metaphase, or anaphase of forage rye, the tiller number per unit area decreased with the postponing of sowing date, and increased with the increase of sowing norm. The sowing date and sowing norm had decisive effect on the number of effective stem per unit area. Suitable population structure was dependent on the increase of effective stem number per plant under the conditions of earlier sowing, and of effective stem number per unit area under the conditions of later sowing.

Regression Analysis↗

[Investigation of daily mitotic activity of the birch, Betula pendula Roth].

A study was made of the daily mitotic activity in the seedling root meristem of birch trees growing in an ecologically clear area--the biological station of Voronezh State University "Venevitinovo". The peak of mitotic activity was exposed at 9 a. m. (according to winter time). The rise of mitotic index was noted at 9 and 12 p. m. due to an increase in the share of cells being in the prophase stage, and to a high number of dividing cells with persistent nucleoli. A possibility of prolongation of the mitotic cycle time is supposed to be due to cell delay in prophase stage, which may be associated with anthropogenic and nature-climatic influences on the original trees themselves and on their seed progeny. This makes it possible to consider the investigated region as only conventionally clear, because of the availability of a high recreative pressure upon the trees.

Betula↗

[A SC35-like protein is localized in the nucleus of Physarum polycephalum].

After being labeled with an anti-SC35 antibody, the specimens of Physarum polycephalum at S, G2, prophase, metaphase and ana-telophase were observed with an Hitachi electron microscope and gold particles marking the location of the SC35-like protein were mainly found in the nucleus,indicating the existence of a SC35-like protein in it. Judging from the densities of the gold particles in the individual domains of the nucleus, the SC35-like protein was principally located in the nucleolar domain and interchromatin domain during G2 and prophase, and the protein was distributed in the interchromosome domain at metaphase and ana-telophase when the nucleus was disintegrated, suggesting that the nucleolus and interchromatin (interchromosome) domain are the two main locations of the SC35-like protein in the nucleus. Further observations upon the nucleolus revealed that the density of the gold particles in the dense fibrillar component (DFC) of the nucleolus was much higher than that of the fibrillar center (FC), demonstrating that the protein was largely situated in the DFC rather than FC.

Animals↗

[Consolidation of cytoskeleton during plant spindle formation. II. "Fused spindles"].

Three mechanisms of fused spindle formation in meiosis of Solanacea have been described: 1) approach of daughter nuclei at prophase II; 2) fusion of perinuclear cytoskeleton systems at prophase II; 3) approach and fusion of prometaphase chaotic figures at prometaphase II. The process of fusion spindle formation appears to be complex and including several steps.

Cytoskeleton↗

Meiotic and sperm chromosome analysis in a male carrier of an inverted insertion (3;10)(q13.2;p14p13).

A phenotypically normal male who fathered a son with the karyotype 46,XY,del(10)(p13) was found to be a balanced carrier of an inverted insertion (3;10) (q13.2;p14p13). Karyotyping five later pregnancies showed four to be unbalanced with respect to the insertion, one of which was also trisomic for chromosome 18. The latest pregnancy was balanced with respect to insertion but had the additional complexity of 47,XXY. In the light of six out of six chromosomally abnormal pregnancies, two of which potentially exhibit an interchromosomal effect, it was decided to investigate the gametic output of the father. Testicular biopsy and semen samples were obtained permitting both meiotic and sperm chromosome analysis. Information was thus obtained at three levels of gamete production, that is, prophase I pairing, chiasma frequency distribution at metaphase I, and sperm karyotypes. Electron microscope studies of synaptonemal complexes showed the rearranged chromosomes to pair fully in meiotic prophase I with no indication of the presence of an insertion. This non-homologous pairing of the inserted region was accompanied by an abnormal frequency distribution of pachytene substages. There was also a reduction in chiasma frequency throughout the genome. However, this did not lead to detectable autosomal univalence or abnormally high X/Y univalence. Thus, the trisomy 18 and XXY pregnancies are unlikely to reflect increased non-disjunctional rates either before or during the first meiotic division. Sperm karyotyping showed that the proportion of chromosomally balanced:unbalanced gametes did not differ from the theoretically expected 1:1. There was no evidence of any increase of unrelated abnormalities in the sperm, further indicating that the overall rate of meiotic non-disjunction was not increased above normal.

Adult↗

Expression of cdc2 and p27(KIP1) phosphorylation in mitotic cells of the human retinoblastoma.

It was recently demonstrated that a lack of p27(KIP1) degradation resulted in the suppression of cdc2 activity and consequent inhibition of entry into the M-phase. The aim of this study was to examine the distribution of phosphorylated p27(KIP1) on threonine 187 (T187-phospho-p27) and cdc2 in mitotic cells of human retinoblastoma, a malignant retinal neoplasm. Several T187-phospho-p27-immunopositive cells were observed in mitotic retinoblastoma cells, but not in the normal retina. Immunoreactivity for T187-phospho-p27 was located in the prophase and metaphase of mitotic tumor cells. In contrast, tumor cells in the anaphase showed no immunoreactivity for T187-phospho-p27. Nuclear expression of cdc2 was detected in many retinoblastoma cells, including mitotic cells. The immunoreactivity in mitotic cells was located in the prophase, as well as metaphase. In contrast, anaphase cells did not show immunoreactivity. Double staining demonstrated the same localization of T187-phospho-p27 and cdc2 in mitotic cells. These results suggest that p27(KIP1) interacts with cdc2 in the M-phase of human retinoblastoma cells.

CDC2 Protein Kinase↗

[Effects of weak light and high temperature stress after anthesis on flag leaf chlorophyll fluorescence and grain fill of wheat].

A field experiment with wheat showed that after anthesis, the photosystem II (PS II), photochemical efficiency (Fv/Fm) and photosynthetic rate (Pn) of flag leaf decreased substantially, its actual quantum yield of PS II electron transport (phiPS II) and photochemical quenching co-efficient (qP) declined slightly, while its non-photochemical quenching (NPQ) increased slightly three days after shading, compared with the control. Three days after high temperature treatment, the Fv/Fm, Pn, phiPS II and qP decreased significantly, while the NPQ increased slightly. During the course of recovery after the stresses, the parameters fluorescence and Pn which were stressed by shading and high temperature at prophase recovered slightly, while those stressed at metaphase decreased all along, indicating that the damage at prophase was reversible, but the stress at metaphase accelerated the caducity of flag leaves. The grain-filling processes of different treatments were modeled with Logistic equation, which showed that the decline of per kernel weight was mainly caused by the decrease of mean filling rate, maximal filling rate, and slow-increase period filling rate. Filling duration, occurrence time of maximal filling rate, duration of slight-increase period, and slight-increase period filling rate were less affected by weak light and high temperature.

Chlorophyll↗

Cytology of Lepidoptera. V. The microtubule cytoskeleton in eupyrene spermatocytes of Ephestia kuehniella (Pyralidae), Inachis io (Nymphalidae), and Orgyia antiqua (Lymantriidae).

The structural transformations of the microtubule cytoskeleton during eupyrene meiosis were characterized in the Mediterranean mealmoth, Ephestia kuehniella. Anti-tubulin immunofluorescence was used in two strains, L and Sbr, of this species. In addition, living primary eupyrene spermatocytes from strain L were observed with phase-contrast light microscopy, and metaphase I spermatocytes were studied with the electron microscope. The analysis did not reveal strain-specific differences in the behavior of the chromatin and the microtubule cytoskeleton in eupyrene meiosis. The general pattern of the two subsequent meiotic divisions in Ephestia kuehniella did not deviate from that observed during nuclear division in other species. A bipolar spindle developed during prophase, chromatin migrated towards the poles in anaphase, and spindle elongation occurred in telophase. Spindle development and behavior were qualitatively almost identical in primary and secondary spermatocytes. The assembly of the second meiotic spindle was initiated during the depolymerization of interzone microtubules of the first division. The relationship between spindle microtubules and centrosomes, however, showed some particularities in the moth. While microtubules radiated out from the spindle poles in late prophase, most microtubules ended about half-way between the equatorial plane and the spindle poles from late prometaphase to early anaphase. The termination of most microtubules distant from the poles was confirmed using electron microscopy in Ephestia kuehniella and using anti-tubulin immunofluorescence in two other Lepidoptera species, Inachis io and Orgyia antiqua. The microtubules were oriented parallel to the pole-to-pole axis of the spindle and were confined to an area laterally sheath by membrane layers. The spindle portions between the pole-proximal termini of the microtubules and the spindle poles contained irregularly shaped membrane elements. In late anaphase, microtubules reform between the migrating chromatin plates and the spindle poles. Concomitantly with the migration of the chromosomes towards the spindle poles, interzone microtubules are assembled. The origin of most of them appears to be at the pole-distal face of the chromatin masses.

Animals↗

[DNA distribution and mitotic index in Ehrlich ascites tumor after colchicine administration].

Additional information like the type of growth is efficient for the interpretation of single parameter DNA distribution curves of tumors. The consideration of mitotic indices leads to further insights. The different growth phases of the Ehrlich ascites tumor are characterized by reproducible DNA histograms. The mitotic index and the frequency of the four mitotic phases turn out to be independent of the growth phase. Colchicine exerts an obvious effect on the prophases and metaphases. In the histogram an accumulation of cells in the G2M phase is evident, also on a higher ploidy level. Nearly identical DNA histograms result in different growth phases. The mitotic index of prophases and metaphases is found to be nearly twice at the 11th and 15th day of growth in comparison with the 8th day. This indicates a higher sensitivity of the Ehrlich ascites tumor to colchicine in steady state which is not expressed in the histogram.

Animals↗

Immunoreactive epidermal growth factor concentrations in follicular fluid obtained from in vitro fertilization.

Immunoreactive epidermal growth factor (EGF) was measured in follicular fluid (FF) obtained at the time of oocyte retrieval for in vitro fertilization from cycles with (91 follicles) and without (128 follicles) the gonadotropin-releasing hormone agonist leuprolide acetate (LA). Follicular fluid immunoreactive EGF levels in the non-LA cycles correlated with androstenedione but not estradiol or progesterone levels from follicles with prophase I oocytes or from all follicles taken together, but not from metaphase I or metaphase II oocyte containing follicles alone. In non-LA cycles, FF immunoreactive EGF levels were lower in follicles that contained metaphase I or II oocytes than prophase I oocytes. Additionally, FF immunoreactive EGF levels were lower in follicles containing metaphase I or II oocytes from non-LA than LA cycles. We conclude that immunoreactive EGF is present in FF. Leuprolide acetate may affect FF immunoreactive EGF levels.

Androstenedione↗

Fine structural studies of early mitotic stages in untreated and nocodazole-treated HeLa cells.

When cells in mitosis are treated with nocodazole, a microtubule-disrupting drug, it can be shown in comparison to untreated cells that microtubules are responsible for the polarized formation of indentations, folds, tubes, and crypts of the nuclear envelope in prophase nuclei. No translocation of chromosomes within the nucleus takes place. Microtubules are not necessary for chromosome condensation, nuclear envelope breakdown, the formation of trilaminar kinetochores, and the orientation of sister-kinetochores within one chromosome in relation to each other. The orientation of kinetochores in relation to the mitotic poles, however, is mediated by microtubules. The data shown here support the working hypothesis about chromosome translocation in prophase nuclei which was presented in an earlier paper.

Benzimidazoles↗

Effects of dimethyl sulphoxide on early gametogenesis in Caenorhabditis elegans: ultrastructural aberrations and loss of synaptonemal complexes from pachytene nuclei.

In Caenorhabditis elegans, loss of viability and fertility was observed after treatment with dimethyl sulphoxide (DMSO). The decrease in life span is associated with senescent morphology of meiotic prophase nuclei, such that nuclei from young and old specimens cannot be differentiated. Aging in oocytes at the pachytene stage of meiotic prophase is characterized by nucleo-cytoplasmic aberrations, increased density of the nucleoplasm and cytoplasm and decrease in numbers of mitochondria (Goldstein and Curis, 1987). Increasing concentrations of DMSO result in decrease in fertility and increased production of abnormal gametes. At DMSO concentrations higher than 5.0%, synaptonemal comlexes (SC) are absent from the nuclei, thus, effective pairing and segregation of homologous chromosomes is not possible. The absence of SCs may be the result of: (1) a premeiotic colchicine-like effect which influences pairing of chromosomes; (2) changes in the structure of the DNA due to DMSO binding that results in changes in expression of the DNA; and (3) changes in temporal DNA synthesis in response to DMSO. Since the SC is essential for regulating pairing and subsequent separation of bivalents, the lack of an SC explains the loss of fertility, due to the production of unbalanced gametes, observed in DMSO treated specimens.

Animals↗

High-resolution R-banding at the 1250-band level. 1. Technical considerations on cell synchronization and R-banding (RHG and RBG).

A comparative analysis of variables for cell synchronization was made and led to the description of optimal conditions capable of yielding, in over 95% of cases, a large number of excellent quality cells in prometaphase and late prophase. Thymidine presents advantages over amethopterin as the synchronizing agent. The block was released with either thymidine or 5-bromo-2'-deoxyuridine (BrdU) for which various concentrations were tested. The presence of colcemid was also evaluated. Without colcemid, the optimal length of the release period was precisely determined so that the wave of synchronized cells could be harvested while going through the early stages of mitoses. Subsequently, GTG, RHG and RBG banding were produced on these elongated chromosomes. A comprehensive approach to RHG banding ensured an easier and well reproducible banding technique. Nine interrelated factors which influence banding quality were studied. The analysis of their effects on this banding pattern revealed new data for the understanding of its mechanism. A high-resolution R-banding technique after BrdU incorporation and Giemsa staining is presented; it is simple, reliable and reproducible. Different conditions for the FPG (Fluorochrome-Photolysis-Giemsa) technique were studied in order to obtain sharper borders and higher contrast between positive and negative bands. Optimal conditions for the incorporation of BrdU and the FPG method produced excellent band separation and band contrast even in very elongated prophase chromosomes. They did not decrease the mitotic index, did not increase chromosomal damage significantly, and did not greatly vary from subject to subject nor with the age of the slide (between 1 day and 36 months). Homologue discordance correlated to band range was compared for GTG, RHG and RBG banding.

Adult↗

[Changes in the surface and cytoskeletal apparatus of the endotheliocytes of the rat aorta during division (based on scanning electron microscopic data)].

Endothelium of the abdominal aorta of 32 KWR-line rats was exposed to freeze injury. En face preparations were made to look for endothelial cells (EC) on different stages of mitosis. Specimens were dried by critical point technique, and the grid was placed on their surface. Then specimens were investigated in succession with light and scanning electron microscopes. The cytoskeleton of EC was investigated on detergent-extracting preparations. It is shown that the end of phase S of the cell cycle and the beginning of prophase are characterized with the lifting of the nuclear-containing zone. Fine microvilli appear on the EC surface during prophase. The cytoskeleton becomes more structured and polarized. During metaphase, EC becomes spherical, its microvilli are shortened. Fine cytoplasmic shoots are seen to extend from the cell poles to the substratum. The density of the fibrillar structures swiftly rise. During anaphase the EC surface is covered with blebs. During late telophase the surface of dividing EC becomes flatter. Their nuclei are connected with rare bundles of fibrillar structures. Mechanisms of EC surface changes during mitosis and the role of cytoskeletal elements are discussed.

Animals↗

Phorbol ester-induced G2 delay in HeLa cells analyzed by time lapse photography.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown previously to mimic X-irradiation in altering cell cycle parameters in Hela cells [Kinzel, V., Richards, J., and Stöhr, M., Science (Wash. DC), 210: 429-431, 1980]. These changes include a delay in G2 phase from which cells recover in the presence of TPA, which suggests an involvement of cellular mediators. In order to obtain information on the onset and the duration of the G2 delay, as well as the onset and rate of recovery, a time-lapse study has been carried out. The analysis of cells in prophase shows that at 10(-6) M and 10(-7) M concentrations, TPA and 12-O-retinoylphorbol-13-acetate (RPA) cause a G2 delay which lasts on the order of 3.5 to 4 h. Below 10(-7) M of RPA and below 10(-8) M of TPA a clear-cut inhibition of HeLa cells in G2 is no longer detectable by this method. These results for a given phorbol ester are dose dependent within a certain range but unlike the case of X-rays are not proportional to dose. Within the dose range studied the recovery rate follows the opposite order. At 10(-6) M TPA and RPA an indication of a parasynchronous burst is observed. At smaller concentrations or with less biological activity of phorbol ester, the cell multiplication rate approaches that of the control or remains even smaller. Possible reasons are discussed. The determination of the transition points seems to indicate that the cellular events inhibited in G2 occur shortly before visible prophase.

Cell Cycle↗