Until a few years ago, it was assumed that oocyte renewal did not take place in the ovary of adult organisms; however, the existence of germline progenitor cells (GPCs), which renew the ovarian follicular reserve, has now been documented in mammals. Specifically, in the adult ovary of bats, the presence of cells located in the cortical region with characteristics similar to GPCs, called adult cortical germ cells (ACGC), has been observed. One of the requirements that a GPC must fulfil is to be able to proliferate mitotically, so the evaluation of cell proliferation in ACGC is of utmost importance in order to be able to relate them to a parental lineage. Currently, there are several methods to determine cell proliferation, including BrdU labelling or the use of endogenous proliferation markers. Thus, the aim of this work was to evaluate the proliferative activity of ACGC in the adult ovary of the bat Artibeus jamaicensis, using different proliferation markers and correlating these with the protein expression of the transcription factor Oct4 and the germ line marker Ddx4. We found that the expression pattern of the proliferation markers BrdU, PCNA, Ki-67 and pH3 occurs at different times of the cell cycle, so co-localization of two or more of these markers allows us to identify proliferating cells. This allowed us to identify ACGC with proliferative capacity in the adult ovary of A. jamaicensis, suggesting that GPCs renew the follicle reserve during the adult life of the organism.

In response to gonadotropins and androgens, testicular cells produce various molecules that control proper proliferation and differentiation of spermatogenic cells through their paracrine and autocrine actions. However, molecules functioning downstream of the hormonal stimulation are poorly understood. Leukaemia inhibitory factor (Lif) is known to maintain the pluripotency of stem cells including embryonic stem cells and primordial germ cells at least in vitro, but its actual roles in vivo remain to be elucidated. To clarify the function of Lif in teleost (medaka) testes, we examined the effects of Lif on spermatogenesis in a newly established cell culture system using a cell line (named Mtp1) derived from medaka testicular somatic cells as feeder cells. We found that addition of baculovirus-produced recombinant medaka Lif to the culture medium or co-culture with Lif-overexpressing Mtp1 cells increased the number of spermatogonia. In situ hybridization and immunohistochemical analyses of the medaka testes showed that mRNAs and proteins of Lif are expressed in spermatogonia and the surrounding Sertoli cells, with higher expression levels in type A (undifferentiated) spermatogonia than in type B (differentiated) spermatogonia. Our findings suggest that Lif regulates spermatogonial cell proliferation in the medaka.

The objective of this study was to assess the influence of nutritional regimens such as adequate feeding, restricted feeding, and underfeeding–refeeding on the follicle growth and development from caprine ovaries. Goats were divided into three different groups (n = 5 per group). For 24 weeks, goats received elephant grass plus concentrate to provide 1.5 (n = 5) and 0.72 (n = 10) times the energy requirements for maintenance of live weight. Underfed goats were subsequently refed for 6 weeks with the diet of the nourished group (1.5 times the energetic requirements of maintenance). Follicular morphology and morphometry, as well as granulosa cells mitotic index were assessed. Ovarian follicles were classified as small or large preantral follicles, or as small or large antral follicles. Ovarian volume was smaller in animals from both underfed and refed groups than in those animals from fed group. Although no difference in the total number of normal follicles was observed among the nutritional groups, underfed animals presented higher percentages of atretic preantral and small antral follicles when compared with fed animals. Large antral follicles from underfed and refed goats presented a lower mitotic index when compared with fed ones. In conclusion, ovaries from goats challenged with prolonged undernutrition will be functionally compromised, which is characterized by atresia of preantral and small antral follicles and decreased mitotic index of large antral follicles. Refeeding those animals will not recover ovarian function to a same level experienced by goats fed a diet with adequate energy requirements.

Trifluralin selectively controls dodder in alfalfa. However, its soil activity dissipates allowing some late season dodder germination. Thiazopyr applied PRE was also found to control dodder selectively in alfalfa. Thiazopyr was more effective when applied in February than in November. Thiazopyr did not control dodder longer into the season than trifluralin in field tests, however it was more effective in greenhouse experiments. The effect of thiazopyr on dodder shoot tips was similar to that of trifluralin, in that both herbicides produced bulbous cells with thickened, globular nuclei, typical of cells treated with herbicides affecting microtubules.

Dinitroaniline herbicides are absorbed readily by roots and emerging shoots, but shoot exposure is more phytotoxic. Translocation within the plant varies by specific herbicide but commonly is minor. Dinitroaniline herbicides injure plants by binding to tubulin, a dimer protein in the ceil that polymerizes to form microtubules (MTs). MTs form the major part of the mitotic apparatus, including spindle fibers, which enable chromosomes to separate during cell division. Dinitroaniline herbicides prevent tubulin from polymerizing into MTs, thus arresting mitosis. This leads to abnormal cells with more than the normal complement of chromosomes and, frequently, lobed nuclei. MTs also are responsible for orienting cell wall microfibrils in such a way that they prevent lateral enlargement of cells. Treatment with dinitroaniline herbicides leads to disorientation of the microfibrils, leading to one of the common symptoms—spherical cells instead of rectangular ones. Studies on the metabolism of trifluralin in plants have shown that amination, dealkylation, and cyclization all can occur. However, metabolites often amount to a small percentage of the original herbicide. In general, trifluralin seems quite stable within the plant.

The herbicidal action of sethoxydim {2-[1-(ethoxyimino)butyl]-5-[2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen-1-one} on corn (Zea mays L. ‘Goldencrossbantam’) was investigated in preemergence and postemergence experiments and hydroponic culture. Soil-applied sethoxydim did not inhibit corn germination. Leaves failed to emerge through the coleoptile with a high herbicide rate (1.6 kg ai/ha), but chlorotic leaves emerged at a lower rate (0.8 kg ai/ha). Growth of corn seedlings treated with a foliar application of 0.2 kg ai/ha was inhibited within 1 day after treatment, but at a lower application rate (0.05 kg ai/ha) growth continued with chlorotic zones on newly expanding leaves. Over the concentration range 3 × 10-5 to 1 × 10-6 M, sethoxydim inhibited the growth of primary roots of corn in hydroponic culture within 24 h. Cytological investigation showed that sethoxydim inhibited cell division but did not interfere with mitosis.

Four days after treatment of corn (Zea mays L. 'SX111′) seedlings with BAS 9052 OH {2 - [1-(ethoxyimino) butyl] - 5 - [2 - (ethylthio) - propyl] - 3 - hydroxy - 2 - cyclohexene-1 - one} growth was inhibited and a purple color developed in the leaves. Low dosages (0.02 and 0.04 kg/ha) caused bleaching of the newly expanding leaves. Dosages higher than 0.04 kg/ha caused significant reductions in length and fresh and dry weights of both shoots and roots. Anthocyanin and total - sugar content increased, whereas chlorophyll accumulation and respiratory activity were reduced by herbicide treatment. BAS 9052 OH did not alter the mitotic index of corn root tips, but induced binucleate cells by preventing cell-plate and -wall formation. In some cells, the daughter nuclei were reoriented with respect to the longitudinal axis of the cells, and in others, the nuclei failed to migrate to the poles. These physiological and cytological effects of BAS 9052 OH on corn seedlings may be responsible for the phytotoxic action of the herbicide on grasses.

The effects of haloxyfop on elongation, mitotic index, and morphology of sorghum and unicorn-plant primary roots were examined. Elongation of sorghum roots was completely inhibited by haloxyfop concentrations of 10–6 M or greater 24 h after treatment, whereas unicorn-plant root elongation was unaffected by the same concentrations 72 h after treatment. Mitotic indices of sorghum roots were reduced by both 10–6 and 10–8 M haloxyfop, with the higher concentration reducing the index to near zero by 24 h of exposure. The mitotic indices of unicorn-plant roots were unaffected by the same levels of the herbicide after both 24- and 48-h treatment. Histological analyses showed that after 24 h exposure to 10–6 M haloxyfop, large vacuoles were present in cells at the root apex that normally did not exhibit these organelles. After 48 h of exposure many cells in the apical region appeared to lack visible cytoplasm and/or nuclei, and by 72 h only cell walls remained visibly evident, and many cells had collapsed. These changes are similar to those that occur in tissues undergoing senescence. Treatment of sorghum roots with 10–8 M haloxyfop did not cause discernible changes after 72 h. Unicorn-plant roots treated with 10–6 M haloxyfop appeared unaffected after 72 h.

Herbicides inhibit biochemical and physiological processes or both with lethal consequences. The target sites of these small molecules are usually enzymes involved in primary metabolic pathways or proteins carrying out essential physiological functions. Herbicides tend to be highly specific for their respective target sites and have served as tools to study these physiological and biochemical processes in plants (Dayan et al. 2010b).

Chagas' disease, caused by the protozoan Trypanosoma cruzi, represents a serious health problem in Latin America, and the available chemotherapy, which is based on 2 nitro-derivatives, is not satisfactory. In folk medicine, natural products including naphthoquinones have been employed for the treatment of different parasitic diseases. In the pursuit of alternative drugs for Chagas' disease, we investigated the mechanism of action of the triazolic naphthoquinone (TN; 2,2-dimethyl-3-(4-phenyl-1H-1,2,3-triazol-1-yl)-2,3-dihydronaphtho[1,2-b]furan-4,5-dione), which is the most active compound against T. cruzi trypomastigotes among a series of naphthofuranquinones. TN was active against the 3 parasite forms producing a dose-dependent inhibitory effect. In epimastigotes, TN induced reservosome disruption, flagellar blebbing, Golgi disorganization, the presence of cytosolic concentric membrane structures and abnormal multiflagellar parasites. The treatment also led to the appearance of well-developed endoplasmic reticulum profiles surrounding organelles that associated with an increase in monodansylcadaverine labelling, suggesting autophagy as part of the TN mechanism of action. Interestingly, no ultrastructural damage was detected in the mitochondria of naphthoquinone-treated epimastigotes. Flow cytometric analysis demonstrated an impairment of mitosis, an increase in ROS production and the maintenance of mitochondrial membrane potential. TN could be a good starting point in the investigation of a chemotherapeutic approach for the treatment of Chagas' disease.

We describe a novel procedure for the immunofluorescent investigation of Plasmodium falciparum. This has allowed us to visualize clearly microtubular structures and their changing conformation through the erythrocytic cell-cycle, to the stage of cytodifferentiation leading to merozoite release. The images of spindle development we observed, together with an analysis of nuclear body numbers in large numbers of parasites, indicate that there is an apparent asynchrony in chromosomal multiplication within a single parasite. Using antibodies specific for post-translational modification of α- tubulin, we also demonstrate that the C-terminal tyrosine-containing epitope of P. falciparum α-tubulin I is similar to that of other organisms. Lysine-40 in the same molecule, a target for highly specific in vivo acetylation in some organisms, is unmodified in the blood stages we examined here. After in vitro acetylation of this residue, however, the epitope to which it contributes was recognized by antibody, showing that the conformation of this part of the molecule is also conserved, despite a lack of primary sequence homology immediately downstream of the target lysine residue.

Isoflavonoids and fish oil may be protective against colorectal cancer, but the evidence in relation to breast cancer risk is ambiguous. In the present study, we have investigated the impact of soya-derived isoflavonoids and n-3 fatty acids from fish oil, both individually and in combination, on apoptosis, cell proliferation and oestrogen receptor (ER) expression in the colon and mammary gland of the rat. Female rats were fed diets high in n-3 fatty acids (80 g/kg diet) or soya protein (765 mg/kg diet isoflavones) for 2 weeks, and then killed before the removal of the colon and mammary glands. Cell proliferation and apoptosis were quantified morphologically in whole crypts and terminal end buds. The expressions of ERα and ERβ were measured in colon tissue scrapes and the mammary gland. Fish oil significantly increased apoptosis and decreased mitosis in both tissues, an effect associated with a decrease in the expressions of ERα and ERβ. Soya had no effect on apoptosis in either tissue, but reduced mitosis in the colon (P < 0·001) while increasing it in the mammary gland (P = 0·001). The changes in proliferation were associated with contrasting changes in the ER expression such that fish oil significantly decreased both ERβ and ERα, while soya increased ERα and decreased ERβ. The results may provide a novel mechanism by which n-3 fatty acids could reduce cancer risk, but the interpretation of the results in relation to soya consumption and breast cancer risk requires further investigation.

We have used confocal microscopy and a fluorescent calmodulin probe to examine the mechanism of localisation of calmodulin during the first cell cycle of the sea urchin zygote. Using fluoresceincalmodulin, calmodulin can be observed within the nucleus and interphase astral microtubule arrays as cells approach mitosis. During mitosis, calmodulin redistributes to the mitotic apparatus and to condensed chromosomes. Quantitative analysis with reference to a control dye (fluorescein-dextran) shows that the distribution of calmodulin is specific. We used a competitive inhibitor of calcium-dependent calmodulin binding (Trp-peptide; Török & Trentham (1994) Biochemistry 33, 12807–20) to test whether the cell cycle localisation of calmodulin was due to its binding to targets on activation. The Trp-peptide eliminates localisation of calmodulin within the nucleus. However, microtubule localisation persists in the presence of the Trp-peptide. These data show that calmodulin can localise by calcium (and hence activation)-dependent as well as calcium-independent mechanisms. This suggests that distinct mechanisms of localisation may be involved in the regulation of the differential functions of calmodulin, at least during the cell cycle.

Spindles were isolated from deposited eggs of the Mediterranean mealmoth, Ephestia Kuehniella. Their structure and development were studied using anti-tubulin immunofluorescence. The microtubules were labelled with three different monoclonal antibodies. These were directed against β-tubulin, tyrosinated α-tubulin and acetylated α-tubulin. Significant differences in the staining behaviour were not detected with the three antibodies. An unusual mode of spindle formation was observed during the first mitotic division after fusion of the pronuclei. Several of the ensuing embryonic divisions may show the same phenomenon. Prophase of these divisions was characterised by an irregular arrangement of microtubules in the nuclear area. The microtubule mass in the nuclear area increased concomitantly with chromosome condensation. Microtubular foci, comparable to the forming asters of canonical spindles, were not detected. The formation of an orderly pattern in the microtubule mass was signalled by the appearance of minispindles apparently developing around individual chromosomes. Several minispindles subsequently aligned and formed metaphase-like entities within the nuclear area. The metaphase-like entities, in turn, aligned with one another and gave rise to a conventional bipolar metaphase spindle with small asters. The further development of the spindle was conventional. The chromosomes migrated towards the spindle poles and finally daughter nuclei formed. The anaphase and telophase spindles possessed both a prominent array of interzone microtubules and asters. The events in prophase of early embryonic mitosis of E. kuehniella may represent a rare case of chromosomeinduced spindle formation.

In spite of its limitations, the culture of Cryptosporidium parvum in monolayers of epithelial cells is a suitable model to study the interaction of this protozoan parasite with the host cell, to assay oocyst infectivity, and to screen drugs for anti-cryptosporidial activity. For unknown reasons, growth of Cryptosporidium in culture is limited in time and generally does not lead to the production of significant numbers of oocysts. In monolayers infected with high doses of oocysts, we observed that many cells remain uninfected, suggesting that some cells are less susceptible to the infection. Since C. parvum and the related species C. hominis lack many essential biosynthetic pathways, we tested whether the dependence of the parasite on host cell metabolites may favour the infection of cells in mitosis. The proportion of monolayer cells in stationary (G0/G1) phase and in mitosis (S/G2/M) was determined and the prevalence of infected cells in each subpopulation was quantified. Although C. parvum infects and develops in dividing and stationary cells, a significant preference for cells in S/G2/M phase was observed. Consistent with previous observations showing that C. parvum induces apoptosis in cell monolayers, infection was accompanied by a significant increase in the proportion of mitotic cells.

The nature, number, structure and behaviour of chromosomes during the meiotic and mitotic sequences in the armoured ground cricket, Acanthoplus speiseri Brancsik, were investigated from testicular and ovariole material. The karyotype of A. speiseri is 2n ♂=25 (24 autosomes + XO) and 2n ♀=26 (24 autosomes + XX). The species exhibits the primitive XO ♂ /XX ♀ type of sex determination mechanism. All chromosomes in the cell complement (including the X sex chromosome), in both sexes, are acrocentric in shape. The X sex chromosome is the largest element in the karyotype while the autosomes comprise three large, four medium and five small pairs. The behaviour of the chromosomes during the meiotic and mitotic processes is normal and typically orthopteran. The study discusses the important role that karyology and cytogenetics can play for species diagnostics in the subfamily Hetrodinae.

The morphological characteristics of mitotic interphase, prophase and metaphase nuclei were analysed for 21 taxa in 14 genera from three different tribes of subfamily Cyrtandroideae (Gesneriaceae) from Asia. Interphase nuclei were classified into three categories: (1) complex chromocentres, as in Ancylostemon, Briggsia, Briggsiopsis, Chirita, Loxostigma, Lysionotus, Oreocharis, Petrocosmea, Raphiocarpus and Whytockia, (2) simple chromocentres, as in Paraboea and Rhabdothamnopsis, and (3) round prochromosomes, as in Anna and Didymocarpus. Species with complex chromocentres seem to be most common among all three tribes. The data further indicate that tribe Didymocarpeae is cytologically much more heterogeneous than other tribes, which accords with the diversity in morphological characters. Three types of chromosome condensation pattern during mitotic prophase were observed, here described as interstitial, gradient and proximal. Except for Ancylostemon, exhibiting a proximal type, and Lysionotus and Whytockia possessing a gradient type, all other species analysed to date exhibit the interstitial type. Our study also reports the chromosome numbers of 12 species in 10 genera of subfamily Cyrtandroideae from China and Vietnam; 11 of these counts are new.

Proliferating cell nuclear antigen (PCNA) plays an essential role in nucleic acid metabolism as a component of the replication and repair machinery. This protein encircles DNA and can slide bidirectionally along the duplex binding to DNA polymerase δ and ε. It is well known that PCNA interacts with proteins involved in the cell cycle. The PCNA interactions with different cellular proteins and the importance of these interactions are discussed. To examine mitotic germinal epithelium proliferation during annual discontinuous spermatogenesis in the lizard Podarcis s. sicula, temporal and the spatial PCNA expression were investigated, and provide a useful endogenous molecular marker.