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Sample records for sperm cells subcellular

  1. Changes in subcellular elemental distributions accompanying the acrosome reaction in sea urchin sperm

    SciTech Connect

    Cantino, M.E.; Schackmann, R.W.; Johnson, D.E.

    1983-05-01

    Energy-dispersive x-ray microanalysis was used to analyze changes in the subcellular distributions of Na, Mg, P, S, Cl, K, and Ca associated with the acrosome reaction of sea urchin sperm. Within 5 sec after induction of the acrosome reaction, nuclear Na and mitochondrial Ca increased and nuclear and mitochondrial K decreased. Uptake of mitochondrial P was detected after several minutes, and increases in nuclear Mg were detected only after 5-10 min of incubation following induction of the reaction. The results suggest that sudden permeability changes in the sperm plasma membrane are associated with the acrosome reaction, but that complete breakdown of membrane and cell function does not occur for several minutes.

  2. Sperm cells manipulation employing dielectrophoresis.

    PubMed

    Rosales-Cruzaley, E; Cota-Elizondo, P A; Sánchez, D; Lapizco-Encinas, Blanca H

    2013-10-01

    Infertility studies are an important growing field, where new methods for the manipulation, enrichment and selection of sperm cells are required. Microfluidic techniques offer attractive advantages such as requirement of low sample volume and short processing times in the range of second or minutes. Presented here is the application of insulator-based dielectrophoresis (iDEP) for the enrichment and separation of mature and spermatogenic cells by employing a microchannel with cylindrical insulating structures with DC electric potentials in the range of 200-1500 V. The results demonstrated that iDEP has the potential to concentrate sperm cells and distinguish between mature and spermatogenic cells by exploiting the differences in shape which lead to differences in electric polarization. Viability assessments revealed that a significant percentage of the cells are viable after the dielectrophoretic treatment, opening the possibility for iDEP to be developed as a tool in infertility studies.

  3. Subcellular localization of phospholipase Cζ in human sperm and its absence in DPY19L2-deficient sperm are consistent with its role in oocyte activation

    PubMed Central

    Escoffier, Jessica; Yassine, Sandra; Lee, Hoi Chang; Martinez, Guillaume; Delaroche, Julie; Coutton, Charles; Karaouzène, Thomas; Zouari, Raoudha; Metzler-Guillemain, Catherine; Pernet-Gallay, Karin; Hennebicq, Sylviane; Ray, Pierre F.; Fissore, Rafael; Arnoult, Christophe

    2015-01-01

    We recently identified the DPY19L2 gene as the main genetic cause of human globozoospermia (70%) and described that Dpy19l2 knockout (KO) mice faithfully reproduce the human phenotype of globozoospermia making it an excellent model to characterize the molecular physiopathology of globozoospermia. Recent case studies on non-genetically characterized men with globozoospermia showed that phospholipase C, zeta (PLCζ), the sperm factor thought to induce the Ca2+ oscillations at fertilization, was absent from their sperm, explaining the poor fertilization potential of these spermatozoa. Since 30% of globozoospermic men remain genetically uncharacterized, the absence of PLCζ in DPY19L2 globozoospermic men remains to be formally established. Moreover, the precise localization of PLCζ and the reasons underlying its loss during spermatogenesis in globozoospermic patients are still not understood. Herein, we show that PLCζ is absent, or its presence highly reduced, in human and mouse sperm with DPY19L2-associated globozoospermia. As a consequence, fertilization with sperm from Dpy19l2 KO mice failed to initiate Ca2+ oscillations and injected oocytes remained arrested at the metaphase II stage, although a few human oocytes injected with DPY19L2-defective sperm showed formation of 2-pronuclei embryos. We report for the first time the subcellular localization of PLCζ in control human sperm, which is along the inner acrosomal membrane and in the perinuclear theca, in the area corresponding to the equatorial region. Because these cellular components are absent in globozoospermic sperm, the loss of PLCζ in globozoospermic sperm is thus consistent and reinforces the role of PLCζ as an oocyte activation factor necessary for oocyte activation. In our companion article, we showed that chromatin compaction during spermiogenesis in Dpy19l2 KO mouse is defective and leads to sperm DNA damage. Together, these defects explain the poor fertilization potential of DPY19L2

  4. Nuclear microscopy of sperm cell elemental structure

    SciTech Connect

    Bench, G.S.

    1994-12-31

    Theories have suggested that there is a link between protamine concentrations in individual sperm and sperm fertility. At present, biochemical analyses have only been performed on bulk populations and existing methods have not been able to determine what percentage of morphologically normal sperm are biochemically defective and potentially infertile. As part of an investigation into male sperm fertility, nuclear microscopy has been utilized to measure elemental profiles at the single sperm level. By measuring the ratio of Phosphorus to Sulfur the authors have been able to determine the amount of protamine 1 and protamine 2 in individual cells from bulk fertile samples of bull and mouse sperm. Preliminary results show that, for each species, the relative amounts of protamine 1 and protamine 2 in morphologically normal sperm agree well with expected values.

  5. Subcellular optogenetics – controlling signaling and single-cell behavior

    PubMed Central

    Karunarathne, W. K. Ajith; O'Neill, Patrick R.; Gautam, Narasimhan

    2015-01-01

    ABSTRACT Variation in signaling activity across a cell plays a crucial role in processes such as cell migration. Signaling activity specific to organelles within a cell also likely plays a key role in regulating cellular functions. To understand how such spatially confined signaling within a cell regulates cell behavior, tools that exert experimental control over subcellular signaling activity are required. Here, we discuss the advantages of using optogenetic approaches to achieve this control. We focus on a set of optical triggers that allow subcellular control over signaling through the activation of G-protein-coupled receptors (GPCRs), receptor tyrosine kinases and downstream signaling proteins, as well as those that inhibit endogenous signaling proteins. We also discuss the specific insights with regard to signaling and cell behavior that these subcellular optogenetic approaches can provide. PMID:25433038

  6. Comparative transcriptomics of Arabidopsis sperm cells.

    PubMed

    Borges, Filipe; Gomes, Gabriela; Gardner, Rui; Moreno, Nuno; McCormick, Sheila; Feijó, José A; Becker, Jörg D

    2008-10-01

    In flowering plants, the two sperm cells are embedded within the cytoplasm of the growing pollen tube and as such are passively transported to the embryo sac, wherein double fertilization occurs upon their release. Understanding the mechanisms and conditions by which male gametes mature and take part in fertilization are crucial goals in the study of plant reproduction. Studies of gene expression in male gametes of maize (Zea mays) and Plumbago and in lily (Lilium longiflorum) generative cells already showed that the previously held view of transcriptionally inert male gametes was not true, but genome-wide studies were lacking. Analyses in the model plant Arabidopsis (Arabidopsis thaliana) were hindered, because no method to isolate sperm cells was available. Here, we used fluorescence-activated cell sorting to isolate sperm cells from Arabidopsis, allowing GeneChip analysis of their transcriptome at a genome-wide level. Comparative analysis of the sperm cell transcriptome with those of representative sporophytic tissues and of pollen showed that sperm has a distinct and diverse transcriptional profile. Functional classifications of genes with enriched expression in sperm cells showed that DNA repair, ubiquitin-mediated proteolysis, and cell cycle progression are overrepresented Gene Ontology categories. Moreover, analysis of the small RNA and DNA methylation pathways suggests that distinct mechanisms might be involved in regulating the epigenetic state of the paternal genome. We identified numerous candidate genes whose involvement in sperm cell development and fertilization can now be directly tested in Arabidopsis. These results provide a roadmap to decipher the role of sperm-expressed proteins.

  7. Sperm Cells of a Primitive Strepsipteran

    PubMed Central

    Nardi, James B.; Delgado, Juan A.; Collantes, Francisco; Miller, Lou Ann; Bee, Charles M.; Kathirithamby, Jeyaraney

    2013-01-01

    The unusual life style of Strepsiptera has presented a long-standing puzzle in establishing its affinity to other insects. Although Strepsiptera share few structural similarities with other insect orders, all members of this order share a parasitic life style with members of two distinctive families in the Coleoptera—the order now considered the most closely related to Strepsiptera based on recent genomic evidence. Among the structural features of several strepsipteran families and other insect families that have been surveyed are the organization of testes and ultrastructure of sperm cells. For comparison with existing information on insect sperm structure, this manuscript presents a description of testes and sperm of a representative of the most primitive extant strepsipteran family Mengenillidae, Eoxenos laboulbenei. We compare sperm structure of E. laboulbenei from this family with that of the three other families of Strepsiptera in the other strepsipteran suborder Stylopidia that have been studied as well as with members of the beetle families Meloidae and Rhipiphoridae that share similar life histories with Strepsiptera. Meloids, Rhipiphorids and Strepsipterans all begin larval life as active and viviparous first instar larvae. This study examines global features of these insects’ sperm cells along with specific ultrastructural features of their organelles. PMID:26462430

  8. Sperm Cells of a Primitive Strepsipteran.

    PubMed

    Nardi, James B; Delgado, Juan A; Collantes, Francisco; Miller, Lou Ann; Bee, Charles M; Kathirithamby, Jeyaraney

    2013-09-04

    The unusual life style of Strepsiptera has presented a long-standing puzzle in establishing its affinity to other insects. Although Strepsiptera share few structural similarities with other insect orders, all members of this order share a parasitic life style with members of two distinctive families in the Coleoptera-the order now considered the most closely related to Strepsiptera based on recent genomic evidence. Among the structural features of several strepsipteran families and other insect families that have been surveyed are the organization of testes and ultrastructure of sperm cells. For comparison with existing information on insect sperm structure, this manuscript presents a description of testes and sperm of a representative of the most primitive extant strepsipteran family Mengenillidae, Eoxenos laboulbenei. We compare sperm structure of E. laboulbenei from this family with that of the three other families of Strepsiptera in the other strepsipteran suborder Stylopidia that have been studied as well as with members of the beetle families Meloidae and Rhipiphoridae that share similar life histories with Strepsiptera. Meloids, Rhipiphorids and Strepsipterans all begin larval life as active and viviparous first instar larvae. This study examines global features of these insects' sperm cells along with specific ultrastructural features of their organelles.

  9. Mechanosensitive subcellular rheostasis drives emergent single-cell mechanical homeostasis

    PubMed Central

    Chen, Weiqiang; Fu, Jianping

    2016-01-01

    Mechanical homeostasis - a fundamental process by which cells maintain stable states under environmental perturbations - is regulated by two subcellular mechanotransducers: cytoskeleton tension and integrin-mediated focal adhesions (FAs)1-5. Here, we show that single-cell mechanical homeostasis is collectively driven by the distinct, graduated dynamics (rheostasis) of subcellular cytoskeleton tension and FAs. Such rheostasis involves a mechanosensitive pattern wherein ground states of cytoskeleton tension and FA determine their distinct reactive paths via either relaxation or reinforcement. Pharmacological perturbations of the cytoskeleton and molecularly modulated integrin catch-slip bonds biased the rheostasis and induced non-homeostasis of FAs, but not of cytoskeleton tension, suggesting a unique sensitivity of FAs in regulating homeostasis. Theoretical modeling revealed myosin-mediated cytoskeleton contractility and catch-slip-bond-like behaviors in FAs and the cytoskeleton as sufficient and necessary mechanisms for quantitatively recapitulating mechanosensitive rheostasis. Our findings highlight previously underappreciated physical nature of the mechanical homeostasis of cells. PMID:27240108

  10. Mechanosensitive subcellular rheostasis drives emergent single-cell mechanical homeostasis

    NASA Astrophysics Data System (ADS)

    Weng, Shinuo; Shao, Yue; Chen, Weiqiang; Fu, Jianping

    2016-09-01

    Mechanical homeostasis--a fundamental process by which cells maintain stable states under environmental perturbations--is regulated by two subcellular mechanotransducers: cytoskeleton tension and integrin-mediated focal adhesions (FAs). Here, we show that single-cell mechanical homeostasis is collectively driven by the distinct, graduated dynamics (rheostasis) of subcellular cytoskeleton tension and FAs. Such rheostasis involves a mechanosensitive pattern wherein ground states of cytoskeleton tension and FA determine their distinct reactive paths through either relaxation or reinforcement. Pharmacological perturbations of the cytoskeleton and molecularly modulated integrin catch-slip bonds biased the rheostasis and induced non-homeostasis of FAs, but not of cytoskeleton tension, suggesting a unique sensitivity of FAs in regulating homeostasis. Theoretical modelling revealed myosin-mediated cytoskeleton contractility and catch-slip-bond-like behaviours in FAs and the cytoskeleton as sufficient and necessary mechanisms for quantitatively recapitulating mechanosensitive rheostasis. Our findings highlight the previously underappreciated physical nature of the mechanical homeostasis of cells.

  11. Comparative Transcriptomics of Arabidopsis thaliana Sperm Cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In flowering plants the two sperm cells are embedded within the cytoplasm of the growing pollen tube and as such are passively transported to the embryo sac, wherein double fertilization occurs upon their release. Understanding the mechanisms and conditions by which male gametes mature and take part...

  12. Metabolic activity of sperm cells: correlation with sperm cell concentration, viability and motility in the rabbit.

    PubMed

    Sabés-Alsina, Maria; Planell, Núria; Gil, Sílvia; Tallo-Parra, Oriol; Maya-Soriano, Maria José; Taberner, Ester; Piles, Miriam; Sabés, Manel; Lopez-Bejar, Manel

    2016-10-01

    The resazurin reduction test (RRT) is a useful technique to assess the metabolic rate of sperm cells. RRT depends on the ability of metabolically active cells to reduce the non-fluorescent dye resazurin to the fluorescent resorufin. The aim of this study was to develop a vital fluorometric method to evaluate metabolic activity of rabbit sperm cells. Twenty-five rabbit males were included in the study. Viability and morphology, motility and metabolic activity were evaluated using an eosin-nigrosin staining, a computer-assisted semen analysis (CASA) and the RRT, respectively. Spearman rank correlation analysis was used to determine the correlation between RRT and semen parameters. After evaluation, a concentration of 10 × 106 sperm cells/ml was selected for further experiments with RRT. No significant correlation was found between the RRT results and the motility parameters. However, after RRT a significant positive correlation between relative fluorescence units and the percentage of alive spermatozoa (r = 0.62; P = 0.001) and a negative one with the percentage of sperm cells with acrosomic abnormalities (r = -0.45; P < 0.05) were detected. The vital assessment of metabolic rate of sperm cells by RRT could provide more information about semen quality than other routine semen analysis, correlating with sperm viability and acrosome status information.

  13. Subcellular characterization of glucose uptake in coronary endothelial cells.

    PubMed

    Gaudreault, N; Scriven, D R L; Laher, I; Moore, E D W

    2008-01-01

    Despite all the evidence linking glucose toxicity to an increased risk of cardiovascular diseases, very little is known about the regulation of glucose uptake in endothelial cells. We have previously reported an asymmetric distribution of the GLUTs (1-5) and SGLT-1 in en face preparations of rat coronary artery endothelia [Gaudreault N., Scriven D.R., Moore E.D., 2004. Characterisation of glucose transporters in the intact coronary artery endothelium in rats: GLUT-2 upregulated by long-term hyperglycaemia. Diabetologia 47(12),2081-2092]. We assessed this time, through immunocytochemistry and wide field fluorescence microscopy coupled to deconvolution, the presence and subcellular distribution of glucose transporters in cultures of human coronary artery endothelial cells (HCAECs). HCAECs express GLUT-1 to 5 and SGLT-1, but their subcellular distribution lacks the luminal/abluminal asymmetry and the proximity to cell-to-cell junctions observed in intact endothelium. To determine the impact of the transporters' distribution on intracellular glucose accumulation, a fluorescent glucose analog (2-NBDG) was used in conjunction with confocal microscopy to monitor uptake in individual cells; the arteries were mounted in an arteriograph chamber with physiological flow rates. The uptake in both preparations was inhibited by cytochalasin-B and d-glucose and stimulated by insulin, but the distribution of the incorporated 2-NBDG mirrored that of the transporters. In HCAEC it was distributed throughout the cell and in the intact arterial endothelium it was restricted to the narrow cytosolic volume adjacent to the cell-to-cell junctions. We suggest that the latter subcellular organization and compartmentalization may facilitate transendothelial transport of glucose in intact coronary artery.

  14. Microscopic analysis of MTT stained boar sperm cells.

    PubMed

    van den Berg, B M

    2015-01-01

    The ability of sperm cells to develop colored formazan by reduction of MTT was used earlier to develop a spectrophotometric assay to determine the viability of sperm cells for several mammalian species. It was the objective of the present study to visualize microscopically the location of the formazan in boar sperm cells. The MTT staining process of boar sperm cells can be divided into a series of morphological events. Incubation of the sperm cells in the presence of MTT resulted after a few min in a diffuse staining of the midpiece of the sperm cells. Upon further incubation the staining of the midpiece became more intense, and gradually the formation of packed formazan granules became more visible. At the same time, a small formazan stained granule appeared medially on the sperm head, which increased in size during further incubation. After incubation for about 1 h the midpiece granules were intensely stained and more clearly distinct as granules, while aggregation of sperm cells occurred. Around 90% of the sperm cells showed these staining events. At the end of the staining the formazan granules have disappeared from both the sperm cells and medium, whereas formazan crystals appeared as thin crystal threads, that became heavily aggregated in the incubation medium. It was concluded that formazan is taken up by lipid droplets in the cytoplasm. Further, the use of the MTT assay to test for sperm viability should be regarded as a qualitative assay, whereas its practical use at artificial insemination (AI) Stations is limited.

  15. Sperm cells as vectors in the production of transgenic animals

    SciTech Connect

    Prince, R.M.

    1993-04-28

    Transgenic animals are used in industry and in biomedical research in order to provide in vivo experimental model systems. Sperm cells have been reported used as vectors in the production of transgenic animals before, however no approach has of yet proven to be successful. Fertilizing eggs with genetically modified sperm would be advantageous in that sperm are readily accessible and stable, and eggs can be fertilized by modified sperm cells in vivo. Recent elucidations regarding the unique manner of DNA packaging in sperm chromatin by protamines has provided us with the insight for developing a method of introducing foreign DNA into sperm which is likely to succeed where others have failed. We have developed a method for mimicking the in vivo system of sperm chromatin toroid subunits in vitro, concentrating these toroids, and fluorescent visualization. Our present work concerns development of a method to successfully deliver DNA across the cell membranes and into the nucleus.

  16. Upward swimming of a sperm cell in shear flow

    NASA Astrophysics Data System (ADS)

    Omori, Toshihiro; Ishikawa, Takuji

    2016-03-01

    Mammalian sperm cells are required to swim over long distances, typically around 1000-fold their own length. They must orient themselves and maintain a swimming motion to reach the ovum, or egg cell. Although the mechanism of long-distance navigation is still unclear, one possible mechanism, rheotaxis, was reported recently. This work investigates the mechanism of the rheotaxis in detail by simulating the motions of a sperm cell in shear flow adjacent to a flat surface. A phase diagram was developed to show the sperm's swimming motion under different shear rates, and for varying flagellum waveform conditions. The results showed that, under shear flow, the sperm is able to hydrodynamically change its swimming direction, allowing it to swim upwards against the flow, which suggests that the upward swimming of sperm cells can be explained using fluid mechanics, and this can then be used to further understand physiology of sperm cell navigation.

  17. The development of cat testicular sperm cryopreservation protocols: Effects of tissue fragments or sperm cell suspension.

    PubMed

    Chatdarong, Kaywalee; Thuwanut, Paweena; Morrell, Jane M

    2016-01-15

    In endangered animals that have been found dead or sterilized for medical reasons, testis is the ultimate source of haploid DNA or sperm. Thus, preservation of testicular sperm may be performed to rescue their genetics. The aim of this study was to evaluate protocols for testicular sperm freezing: as tissue fragments or cell suspension in domestic cats as a model. A pair of testes from each cat (n = 9) were cut into eight equal pieces. Four randomly selected pieces were cryopreserved as: (1) tissue pieces using two-step freezing; (2) tissue pieces using a slow passive cooling device (CoolCell); (3) sperm suspension after single-layer centrifugation (SLC) through colloids; and (4) sperm suspension without being processed through SLC. A testicular piece from each cat served as fresh control. Testicular sperm membrane and DNA integrity were evaluated before, and after, the cryopreservation process. In addition, spermatogenic cell types (testicular sperm, spermatogonia, spermatocyte, and spermatid) present in the suspension samples were counted before and after SLC. The results found that testicular sperm membrane integrity in the suspension after SLC process was higher than that in the fragment form neither using the two-step nor CoolCell freezing, both before and after freezing (before freezing: 92.3 ± 3.4 vs. 81 ± 4.5 and 80.0 ± 7.0; after freezing: 84.5 ± 4.6 vs. 71.2 ± 12 and 76.2 ± 4.6; P ≤ 0.05). Testicular sperm DNA integrity was, however, not different among groups. Furthermore, the samples processed through the SLC had higher ration of sperm cells: other spermatogenic cells than those were not processed through the SLC (88.9 ± 3.8 vs. 30 ± 7.9; P ≤ 0.05). In summary, testicular sperm cryopreserved as a minced suspension is considered suitable in terms of preventing sperm membrane integrity, and SLC is considered a selection tool for enriching haploid sperm cells from castrated or postmortem cats.

  18. Reliable single sperm cryopreservation in Cell Sleepers for azoospermia management.

    PubMed

    Coetzee, K; Ozgur, K; Berkkanoglu, M; Bulut, H; Isikli, A

    2016-03-01

    Conventional sperm freezing methods perform best when freezing sperm samples containing at least hundreds of spermatozoa. In this severe male factor infertility case series, we examined the reproductive outcomes in 12 intracytoplasmic sperm injection cases where spermatozoa used were frozen in Cell Sleepers. Cell Sleepers are novel devices in which individual spermatozoa can be frozen in microdroplets. The case series included five men with obstructive azoospermia, six with nonobstructive azoospermia and one with cryptozoospermia, in whom microscopic sperm retrievals from testicular sperm extraction (TESE), micro-TESE extracts and a centrifugation procedure resulted in less than 50 spermatozoa. A total of 304 microscopically retrieved spermatozoa were frozen in 20 Cell Sleepers using a rapid manual cryopreservation method. A total of 179 mature oocytes were injected with recovered thawed spermatozoa, resulting in a fertilisation rate of 65.9% (118 of 179), with no total fertilisation failures. In 10 cases, an embryo transfer was performed, three on day 3 and seven on day 5, resulting in a per cycle pregnancy rate of 58.3% (seven of 12). Four of the pregnancies have progressed past 20 gestation weeks. The recovery and use of spermatozoa that were frozen in Cell Sleepers was uncomplicated and effective and eliminated the need to perform any microscopic sperm retrieval procedures on the day of oocyte collection. Modification of the routine sperm cryopreservation methodology to include the use of Cell Sleepers increases the range of sperm samples that can be effectively cryopreserved, to include men with severe male factor fertility.

  19. Nuclear microscopy of sperm cell elemental structure

    NASA Astrophysics Data System (ADS)

    Bench, Graham S.; Balhorn, Rod; Friz, Alexander M.

    1995-05-01

    Theories suggest there is a link between protamine concentrations in individual sperm and male fertility. Previously, biochemical analyses have used pooled samples containing millions of sperm to determine protamine concentrations. These methods have not been able to determine what percentage of morphologically normal sperm are biochemically defective and potentially infertile. Nuclear microscopy has been utilized to measure elemental profiles at the single sperm level. By measuring the amount of phosphorus and sulfur, the total DNA and protamine content in individual sperm from fertile bull and mouse semen have been determined. These values agree with results obtained from other biochemical analyses. Nuclear microscopy shows promise for measuring elemental profiles in the chromatin of individual sperm. The technique may be able to resolve theories regarding the importance of protamines to male fertility and identify biochemical defects responsible for certain types of male infertility.

  20. Nuclear microscopy of sperm cell elemental structure

    SciTech Connect

    Bench, G.S.; Balhorn, R.; Friz, A.M.; Freeman, S.P.H.T.

    1994-09-28

    Theories suggest there is a link between protamine concentrations in individual sperm and male fertility. Previously, biochemical analyses have used pooled samples containing millions of sperm to determine protamine concentrations. These methods have not been able to determine what percentage of morphologically normal sperm are biochemically defective and potentially infertile. Nuclear microscopy has been utilized to measure elemental profiles at the single sperm level. By measuring the amount of phosphorus and sulfur, the total DNA and protamine content in individual sperm from fertile bull and mouse semen have been determined. These values agree with results obtained from other biochemical analyses. Nuclear microscopy shows promise for measuring elemental profiles in the chromatin of individual sperm. The technique may be able to resolve theories regarding the importance of protamines to male fertility and identify biochemical defects responsible for certain types of male infertility.

  1. Subcellular glucose exposure biases the spatial distribution of insulin granules in single pancreatic beta cells

    NASA Astrophysics Data System (ADS)

    Terao, Kyohei; Gel, Murat; Okonogi, Atsuhito; Fuke, Ariko; Okitsu, Teru; Tada, Takashi; Suzuki, Takaaki; Nagamatsu, Shinya; Washizu, Masao; Kotera, Hidetoshi

    2014-02-01

    In living tissues, a cell is exposed to chemical substances delivered partially to its surface. Such a heterogeneous chemical environment potentially induces cell polarity. To evaluate this effect, we developed a microfluidic device that realizes spatially confined delivery of chemical substances at subcellular resolution. Our microfluidic device allows simple setup and stable operation for over 4 h to deliver chemicals partially to a single cell. Using the device, we showed that subcellular glucose exposure triggers an intracellular [Ca2+] change in the β-cells. In addition, the imaging of a cell expressing GFP-tagged insulin showed that continuous subcellular exposure to glucose biased the spatial distribution of insulin granules toward the site where the glucose was delivered. Our approach illustrates an experimental technique that will be applicable to many biological experiments for imaging the response to subcellular chemical exposure and will also provide new insights about the development of polarity of β-cells.

  2. Hydrodynamics of Sperm Cells near Surfaces

    PubMed Central

    Elgeti, Jens; Kaupp, U. Benjamin; Gompper, Gerhard

    2010-01-01

    Sperm are propelled by an actively beating tail, and display a wide variety of swimming patterns. When confined between two parallel walls, sperm swim either in circles or on curvilinear trajectories close to the walls. We employ mesoscale hydrodynamics simulations in combination with a mechanical sperm model to study the swimming behavior near walls. The simulations show that sperm become captured at the wall due to the hydrodynamic flow fields which are generated by the flagellar beat. The circular trajectories are determined by the chiral asymmetry of the sperm shape. For strong (weak) chirality, sperm swim in tight (wide) circles, with the beating plane of the flagellum oriented perpendicular (parallel) to the wall. For comparison, we also perform simulations based on a local anisotropic friction of the flagellum. In this resistive force approximation, surface adhesion and circular swimming patterns are obtained as well. However, the adhesion mechanism is now due to steric repulsion, and the orientation of the beating plane is different. Our model provides a theoretical framework that explains several distinct swimming behaviors of sperm near and far from a wall. Moreover, the model suggests a mechanism by which sperm navigate in a chemical gradient via a change of their shape. PMID:20712984

  3. Raman spectroscopy of DNA packaging in individual human sperm cells distinguishes normal from abnormal cells.

    PubMed

    Huser, Thomas; Orme, Christine A; Hollars, Christopher W; Corzett, Michele H; Balhorn, Rod

    2009-05-01

    Healthy human males produce sperm cells of which about 25-40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with male infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro-Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape. We show that Raman spectra of individual sperm cells contain vibrational marker modes that can be used to assess the efficiency of DNA-packaging for each cell. Raman spectra obtained from sperm cells with normal shape provide evidence that DNA in these sperm is very efficiently packaged. We find, however, that the relative protein content per cell and DNA packaging efficiencies are distributed over a relatively wide range for sperm cells with both normal and abnormal shape. These findings indicate that single cell Raman spectroscopy should be a valuable tool in assessing the quality of sperm cells for in-vitro fertilization.

  4. Raman Spectroscopy of DNA Packaging in Individual Human Sperm Cells distinguishes Normal from Abnormal Cells

    SciTech Connect

    Huser, T; Orme, C; Hollars, C; Corzett, M; Balhorn, R

    2009-03-09

    Healthy human males produce sperm cells of which about 25-40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with male infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro-Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape. We show that Raman spectra of individual sperm cells contain vibrational marker modes that can be used to assess the efficiency of DNA-packaging for each cell. Raman spectra obtained from sperm cells with normal shape provide evidence that DNA in these sperm is very efficiently packaged. We find, however, that the relative protein content per cell and DNA packaging efficiencies are distributed over a relatively wide range for sperm cells with both normal and abnormal shape. These findings indicate that single cell Raman spectroscopy should be a valuable tool in assessing the quality of sperm cells for in-vitro fertilization.

  5. Shape and shear guide sperm cells spiraling upstream

    NASA Astrophysics Data System (ADS)

    Kantsler, Vasily; Dunkel, Jorn; Goldstein, Raymond E.

    2014-11-01

    A major puzzle in biology is how mammalian sperm determine and maintain the correct swimming direction during the various phases of the sexual reproduction process. Currently debated mechanisms for sperm long range travel vary from peristaltic pumping to temperature sensing (thermotaxis) and direct response to fluid flow (rheotaxis), but little is known quantitatively about their relative importance. Here, we report the first quantitative experimental study of mammalian sperm rheotaxis. Using microfluidic devices, we investigate systematically the swimming behavior of human and bull sperm over a wide range of physiologically relevant shear rates and viscosities. Our measurements show that the interplay of fluid shear, steric surface-interactions and chirality of the flagellar beat leads to a stable upstream spiraling motion of sperm cells, thus providing a generic and robust rectification mechanism to support mammalian fertilization. To rationalize these findings, we identify a minimal mathematical model that is capable of describing quantitatively the experimental observations.

  6. Sub-cellular force microscopy in single normal and cancer cells.

    PubMed

    Babahosseini, H; Carmichael, B; Strobl, J S; Mahmoodi, S N; Agah, M

    2015-08-07

    This work investigates the biomechanical properties of sub-cellular structures of breast cells using atomic force microscopy (AFM). The cells are modeled as a triple-layered structure where the Generalized Maxwell model is applied to experimental data from AFM stress-relaxation tests to extract the elastic modulus, the apparent viscosity, and the relaxation time of sub-cellular structures. The triple-layered modeling results allow for determination and comparison of the biomechanical properties of the three major sub-cellular structures between normal and cancerous cells: the up plasma membrane/actin cortex, the mid cytoplasm/nucleus, and the low nuclear/integrin sub-domains. The results reveal that the sub-domains become stiffer and significantly more viscous with depth, regardless of cell type. In addition, there is a decreasing trend in the average elastic modulus and apparent viscosity of the all corresponding sub-cellular structures from normal to cancerous cells, which becomes most remarkable in the deeper sub-domain. The presented modeling in this work constitutes a unique AFM-based experimental framework to study the biomechanics of sub-cellular structures.

  7. Identifying subcellular protein localization with fluorescent protein fusions after transient expression in onion epidermal cells.

    PubMed

    Nebenführ, Andreas

    2014-01-01

    Most biochemical functions of plant cells are carried out by proteins which act at very specific places within these cells, for example, within different organelles. Identifying the subcellular localization of proteins is therefore a useful tool to narrow down the possible functions that a novel or unknown protein may carry out. The discovery of genetically encoded fluorescent markers has made it possible to tag specific proteins and visualize them in vivo under a variety of conditions. This chapter describes a simple method to use transient expression of such fluorescently tagged proteins in onion epidermal cells to determine their subcellular localization relative to known markers.

  8. Interaction of resident sperm with sperm-storage tubule (SST) epithelial cell microvilli in the turkey breeder hen

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Interaction of resident sperm with sperm-storage tubule (SST) epithelial cell microvilli in the turkey breeder hen M.R. Bakst*1 and C. Murphy2, 1Animal Biosciences and Biotechnology Laboratory, 2Electron & Confocal Microscopy Unit, Beltsville Area, ARS, USDA, Beltsville MD Sustained fertilization o...

  9. Probing cell mechanics with subcellular laser dissection of actomyosin networks in the early developing Drosophila embryo.

    PubMed

    Rauzi, M; Lenne, P-F

    2015-01-01

    Laser dissection is a useful tool in developmental biology to probe mechanical forces from the subcellular to the tissue/embryo scale. During tissue morphogenesis, cells are equipped with networks of actomyosin that generate forces. Here we present a technique based on near-infrared (NIR) femtosecond (fs) pulsed laser dissection that allows subcellular ablation of actomyosin networks. This technique allows to selectively ablate actomyosin networks while preserving cell plasma membrane. The resulting relaxation of the remaining network after laser dissection is imaged and analyzed to deduce local forces responsible for tissue morphogenesis in the developing Drosophila embryo.

  10. Geometrical guidance and trapping transition of human sperm cells

    NASA Astrophysics Data System (ADS)

    Guidobaldi, A.; Jeyaram, Y.; Berdakin, I.; Moshchalkov, V. V.; Condat, C. A.; Marconi, V. I.; Giojalas, L.; Silhanek, A. V.

    2014-03-01

    The guidance of human sperm cells under confinement in quasi-2D microchambers is investigated using a purely physical method to control their distribution. Transport property measurements and simulations are performed with diluted sperm populations, for which effects of geometrical guidance and concentration are studied in detail. In particular, a trapping transition at convex angular wall features is identified and analyzed. We also show that highly efficient microratchets can be fabricated by using curved asymmetric obstacles to take advantage of the spermatozoa specific swimming strategy.

  11. Sub-cellular force microscopy in single normal and cancer cells

    SciTech Connect

    Babahosseini, H.; Carmichael, B.; Strobl, J.S.; Mahmoodi, S.N.; Agah, M.

    2015-08-07

    This work investigates the biomechanical properties of sub-cellular structures of breast cells using atomic force microscopy (AFM). The cells are modeled as a triple-layered structure where the Generalized Maxwell model is applied to experimental data from AFM stress-relaxation tests to extract the elastic modulus, the apparent viscosity, and the relaxation time of sub-cellular structures. The triple-layered modeling results allow for determination and comparison of the biomechanical properties of the three major sub-cellular structures between normal and cancerous cells: the up plasma membrane/actin cortex, the mid cytoplasm/nucleus, and the low nuclear/integrin sub-domains. The results reveal that the sub-domains become stiffer and significantly more viscous with depth, regardless of cell type. In addition, there is a decreasing trend in the average elastic modulus and apparent viscosity of the all corresponding sub-cellular structures from normal to cancerous cells, which becomes most remarkable in the deeper sub-domain. The presented modeling in this work constitutes a unique AFM-based experimental framework to study the biomechanics of sub-cellular structures. - Highlights: • The cells are modeled as a triple-layered structure using Generalized Maxwell model. • The sub-domains include membrane/cortex, cytoplasm/nucleus, and nuclear/integrin. • Biomechanics of corresponding sub-domains are compared among normal and cancer cells. • Viscoelasticity of sub-domains show a decreasing trend from normal to cancer cells. • The decreasing trend becomes most significant in the deeper sub-domain.

  12. Autometallographic demonstration of zinc ions in rat sperm cells.

    PubMed

    Stoltenberg, M; Sørensen, M B; Danscher, G; Juhl, S; Andreasen, A; Ernst, E

    1997-09-01

    An in-vitro technique for autometallographic (AMG) demonstration of chelatable zinc in electroejaculated sperm cells and spermatozoa from the epididymis is presented and the localization of zinc ions in rat spermatozoa is described. Sperm cells from caput epididymis showed zinc staining in all parts of the tail and a sparse, dispersed staining in the acrosome. Spermatozoa from cauda epididymis showed heavy staining in the acrosome but no staining in the tail, or post-acrosomal part of the sperm head. This distinct acrosomal AMG staining was also found in ejaculated spermatozoa, but additionally a segmentation of the tail was seen based on differences in staining intensity. The membrane penetrating chelator diethyldithiocarbamate (DEDTC) was found to block the AMG staining whereas calcium-EDTA, known not to pass through cell membranes, did not influence the staining, proving that the detected zinc ions are intracellularly located. Two different approaches for demonstrating the presence of a chelatable zinc pool at electron microscope levels are presented, and the ultrastructural presence of AMG grains located in the acrosome and in the mitochondria of the midpiece is demonstrated. It is postulated that an exchange of zinc ions takes place between the epididymal epithelium and the sperm cells as they pass along the epididymal duct.

  13. Origination of turbulence in dense suspensions of sperm cells

    NASA Astrophysics Data System (ADS)

    Denissenko, Petr; Kirkman-Brown, Jackson; Smith, David; Kantsler, Vasily

    2014-11-01

    Motile micro-organisms with pushing flagella, such as sperm cells, can be directed by ``one way'' microchannels with ratchet teeth-like wall configuration. We use an array of such micro-channels to gradually concentrate human spermatozoa in a circular arena of 1 mm diameter and 200 micron depth. Velocities of individual cells are measured by particle tracking and velocity of cell-carrying fluid is measured using PIV. At high concentrations, fluid velocities and the velocity fluctuations of individual cells exceeding that of individual swimmers in the dilute regime by an order of magnitude have been measured. Velocity correlations are calculated to study evolution of characteristic length scales as the cell concentration increases. Results are discussed in the context of self-organisation phenomena in active fluids and cooperation of sperm cells.

  14. Emergent cell and tissue dynamics from subcellular modeling of active biomechanical processes

    NASA Astrophysics Data System (ADS)

    Sandersius, S. A.; Weijer, C. J.; Newman, T. J.

    2011-08-01

    Cells and the tissues they form are not passive material bodies. Cells change their behavior in response to external biochemical and biomechanical cues. Behavioral changes, such as morphological deformation, proliferation and migration, are striking in many multicellular processes such as morphogenesis, wound healing and cancer progression. Cell-based modeling of these phenomena requires algorithms that can capture active cell behavior and their emergent tissue-level phenotypes. In this paper, we report on extensions of the subcellular element model to model active biomechanical subcellular processes. These processes lead to emergent cell and tissue level phenotypes at larger scales, including (i) adaptive shape deformations in cells responding to slow stretching, (ii) viscous flow of embryonic tissues, and (iii) streaming patterns of chemotactic cells in epithelial-like sheets. In each case, we connect our simulation results to recent experiments.

  15. Detection of dilute sperm samples using photoacoustic flowmetry

    NASA Astrophysics Data System (ADS)

    Viator, J. A.; Sutovsky, P.; Weight, R. M.

    2008-02-01

    Detection of sperm cells in dilute samples may have application in forensic testing and diagnosis of male reproductive health. Due to the optically dense subcellular structures in sperm cells, irradiation by nanosecond laser pulses induces a photoacoustic response detectable using a custom flow cytometer. We determined the detection threshold of bull sperm using various concentrations, from 200 to 1,000,000 sperm cells per milliliter. Using a tunable laser system set to 450nm with a 5 ns pulse duration and 11-12 mJ/pulse, we obtained a detection threshold of 3 sperm cells. The flow rate was 4 ml/minute through the flow chamber. The acoustic sensor was a 100 μm PVDF film attached to the glass flow chamber. The acoustic signal was preamplified and sent to an oscilloscope. The threshold signal indicated a signal to noise ratio of approximately 6 to 1. Improved system design may decrease the threshold to single sperm cells.

  16. In vitro human immunodeficiency virus and sperm cell interaction mediated by the mannose receptor.

    PubMed

    Cardona-Maya, Walter; Velilla, Paula A; Montoya, Carlos Julio; Cadavid, Ángela; Rugeles, María T

    2011-12-01

    Leukocytes are considered to be the main source of HIV-1 infection in semen. However, HIV-1 interaction with spermatozoa has also been demonstrated, suggesting that both spermatozoa and leukocytes might play a role during sexual transmission of HIV-1. The purpose of the present study was to evaluate if HIV-1 particles interact with sperm cells through the mannose receptor (MR), and then to determine the ability of "infected" sperm cells to transmit the virus to susceptible targets. The expression of classical HIV-1 receptor and co-receptors and the MR by sperm cells was determined by flow cytometry; the interaction in vitro between sperm and HIV-1 was evaluated by fluorescence microscopy. Additionally, the in vitro interaction of sperm cells and HIV-1 was determined detecting viral nucleic acids by PCR. D-Mannose was used to block HIV-1-sperm cell interaction. Sperm cells preincubated with HIV-1 particles and activated mononuclear cells were co-cultured to determine viral transmission. The presence of viral RNA was detected in 28% of the samples in which sperm cells were preincubated with HIV-1 particles. Mannose was able to block interaction in 75% of the cases. Finally, we demonstrated that "infected" sperm cells were able to transmit the HIV-1 infection to susceptible targets. In conclusion, these results indicate that the MR is involved in sperm cell-HIV-1 interaction. Our results also suggest that sperm cells could be an important source of infection.

  17. Olfactory receptors are displayed on dog mature sperm cells

    PubMed Central

    1993-01-01

    Olfactory receptors constitute a huge family of structurally related G protein-coupled receptors, with up to a thousand members expected. We have shown previously that genes belonging to this family were expressed in the male germ line from both dog and human. The functional significance of this unexpected site of expression was further investigated in the present study. We demonstrate that a few dog genes representative of various subfamilies of olfactory receptors are expressed essentially in testis, with little or no expression in olfactory mucosa. Other randomly selected members of the family show the expected site of expression, restricted to the olfactory system. Antibodies were generated against the deduced amino acid sequence of the most abundantly expressed olfactory receptor gene in dog testis. The purified serum was able to detect the gene product (DTMT receptor) in late round and elongated spermatids, as well as in the cytoplasmic droplet that characterizes the maturation of dog sperm cells, and on the tail midpiece of mature spermatozoa. Western blotting further confirmed the presence of a 40-kD immunoreactive protein in the membrane of mature sperm cells. Altogether , these results demonstrate that the main expression site of a subset of the large olfactory receptor gene family is not olfactory mucosa but testis. This expression correlates with the presence of the corresponding protein during sperm cell maturation, and on mature sperm cells. The pattern of expression is consistent with a role as sensor for unidentified chemicals possibly involved in the control of mammalian sperm maturation, migration, and/or fertilization. PMID:8253843

  18. Subcellular compartmentalization of saccharide moieties in cultured normal and malignant cells

    PubMed Central

    1980-01-01

    We studied subcellular localization of saccharide moieties in cultured normal and malignant cells fixed in paraformaldehyde and treated with a nonionic detergent, using lectins specific for various surgar residues as probes in fluorescence microscopy. In normal cells, concanavalin A and Lens culinaris agglutinin, specific for mannose-rich carbohydrate cores in glycoproteins, labeled the endoplasmic reticulum as a wide perinuclear region. Other lectins, on the other hand, stained the Golgi apparatus as a juxtanuclear reticular structure. A similar compartmentalization was also seen in all malignant cells studied, although the Golgi apparatus in these cells was distinctly vesicular in appearance. Our results indicate that saccharide moieties in both normal and malignant cells are similarly compartmentalized, and thus speak in favor of a unidirectional subcellular flow for both membrane and secreted glycoconjugates. PMID:7372714

  19. Subcellular compartmentalization of saccharide moieties in cultured normal and malignant cells.

    PubMed

    Virtanen, I; Ekblom, P; Laurila, P

    1980-05-01

    We studied subcellular localization of saccharide moieties in cultured normal and malignant cells fixed in paraformaldehyde and treated with a nonionic detergent, using lectins specific for various surgar residues as probes in fluorescence microscopy. In normal cells, concanavalin A and Lens culinaris agglutinin, specific for mannose-rich carbohydrate cores in glycoproteins, labeled the endoplasmic reticulum as a wide perinuclear region. Other lectins, on the other hand, stained the Golgi apparatus as a juxtanuclear reticular structure. A similar compartmentalization was also seen in all malignant cells studied, although the Golgi apparatus in these cells was distinctly vesicular in appearance. Our results indicate that saccharide moieties in both normal and malignant cells are similarly compartmentalized, and thus speak in favor of a unidirectional subcellular flow for both membrane and secreted glycoconjugates.

  20. Conserved roles of the prion protein domains on subcellular localization and cell-cell adhesion.

    PubMed

    Solis, Gonzalo P; Radon, Yvonne; Sempou, Emily; Jechow, Katharina; Stuermer, Claudia A O; Málaga-Trillo, Edward

    2013-01-01

    Analyses of cultured cells and transgenic mice expressing prion protein (PrP) deletion mutants have revealed that some properties of PrP -such as its ability to misfold, aggregate and trigger neurotoxicity- are controlled by discrete molecular determinants within its protein domains. Although the contributions of these determinants to PrP biosynthesis and turnover are relatively well characterized, it is still unclear how they modulate cellular functions of PrP. To address this question, we used two defined activities of PrP as functional readouts: 1) the recruitment of PrP to cell-cell contacts in Drosophila S2 and human MCF-7 epithelial cells, and 2) the induction of PrP embryonic loss- and gain-of-function phenotypes in zebrafish. Our results show that homologous mutations in mouse and zebrafish PrPs similarly affect their subcellular localization patterns as well as their in vitro and in vivo activities. Among PrP's essential features, the N-terminal leader peptide was sufficient to drive targeting of our constructs to cell contact sites, whereas lack of GPI-anchoring and N-glycosylation rendered them inactive by blocking their cell surface expression. Importantly, our data suggest that the ability of PrP to homophilically trans-interact and elicit intracellular signaling is primarily encoded in its globular domain, and modulated by its repetitive domain. Thus, while the latter induces the local accumulation of PrPs at discrete punctae along cell contacts, the former counteracts this effect by promoting the continuous distribution of PrP. In early zebrafish embryos, deletion of either domain significantly impaired PrP's ability to modulate E-cadherin cell adhesion. Altogether, these experiments relate structural features of PrP to its subcellular distribution and in vivo activity. Furthermore, they show that despite their large evolutionary history, the roles of PrP domains and posttranslational modifications are conserved between mouse and zebrafish.

  1. SubCellProt: predicting protein subcellular localization using machine learning approaches.

    PubMed

    Garg, Prabha; Sharma, Virag; Chaudhari, Pradeep; Roy, Nilanjan

    2009-01-01

    High-throughput genome sequencing projects continue to churn out enormous amounts of raw sequence data. However, most of this raw sequence data is unannotated and, hence, not very useful. Among the various approaches to decipher the function of a protein, one is to determine its localization. Experimental approaches for proteome annotation including determination of a protein's subcellular localizations are very costly and labor intensive. Besides the available experimental methods, in silico methods present alternative approaches to accomplish this task. Here, we present two machine learning approaches for prediction of the subcellular localization of a protein from the primary sequence information. Two machine learning algorithms, k Nearest Neighbor (k-NN) and Probabilistic Neural Network (PNN) were used to classify an unknown protein into one of the 11 subcellular localizations. The final prediction is made on the basis of a consensus of the predictions made by two algorithms and a probability is assigned to it. The results indicate that the primary sequence derived features like amino acid composition, sequence order and physicochemical properties can be used to assign subcellular localization with a fair degree of accuracy. Moreover, with the enhanced accuracy of our approach and the definition of a prediction domain, this method can be used for proteome annotation in a high throughput manner. SubCellProt is available at www.databases.niper.ac.in/SubCellProt.

  2. Rheotaxis facilitates upstream navigation of mammalian sperm cells

    PubMed Central

    Kantsler, Vasily; Dunkel, Jörn; Blayney, Martyn; Goldstein, Raymond E

    2014-01-01

    A major puzzle in biology is how mammalian sperm maintain the correct swimming direction during various phases of the sexual reproduction process. Whilst chemotaxis may dominate near the ovum, it is unclear which cues guide spermatozoa on their long journey towards the egg. Hypothesized mechanisms range from peristaltic pumping to temperature sensing and response to fluid flow variations (rheotaxis), but little is known quantitatively about them. We report the first quantitative study of mammalian sperm rheotaxis, using microfluidic devices to investigate systematically swimming of human and bull sperm over a range of physiologically relevant shear rates and viscosities. Our measurements show that the interplay of fluid shear, steric surface-interactions, and chirality of the flagellar beat leads to stable upstream spiralling motion of sperm cells, thus providing a generic and robust rectification mechanism to support mammalian fertilisation. A minimal mathematical model is presented that accounts quantitatively for the experimental observations. DOI: http://dx.doi.org/10.7554/eLife.02403.001 PMID:24867640

  3. Apparatus and method for measuring single cell and sub-cellular photosynthetic efficiency

    DOEpatents

    Davis, Ryan Wesley; Singh, Seema; Wu, Huawen

    2013-07-09

    Devices for measuring single cell changes in photosynthetic efficiency in algal aquaculture are disclosed that include a combination of modulated LED trans-illumination of different intensities with synchronized through objective laser illumination and confocal detection. Synchronization and intensity modulation of a dual illumination scheme were provided using a custom microcontroller for a laser beam block and constant current LED driver. Therefore, single whole cell photosynthetic efficiency, and subcellular (diffraction limited) photosynthetic efficiency measurement modes are permitted. Wide field rapid light scanning actinic illumination is provided for both by an intensity modulated 470 nm LED. For the whole cell photosynthetic efficiency measurement, the same LED provides saturating pulses for generating photosynthetic induction curves. For the subcellular photosynthetic efficiency measurement, a switched through objective 488 nm laser provides saturating pulses for generating photosynthetic induction curves. A second near IR LED is employed to generate dark adapted states in the system under study.

  4. Cell dipole behaviour revealed by ECM sub-cellular geometry

    NASA Astrophysics Data System (ADS)

    Mandal, Kalpana; Wang, Irène; Vitiello, Elisa; Orellana, Laura Andreina Chacòn; Balland, Martial

    2014-12-01

    Cells sense and respond to their mechanical environment by exerting forces on their surroundings. The way forces are modulated by extra-cellular matrix (ECM) properties plays a key role in tissue homoeostasis. Using highly resolved micropatterns that constrain cells into the same square envelope but vary the adhesive geometry, here we investigate how the adhesive micro-environment affects the architecture of actin cytoskeleton and the orientation of traction forces. Our data demonstrate that local adhesive changes can trigger orientational ordering of stress fibres throughout the cell, suggesting that cells are capable of integrating information on ECM geometry at the whole-cell level. Finally, we show that cells tend to generate highly polarized force pattern, that is, unidirectional pinching, in response to adequate adhesive conditions. Hence, the geometry of adhesive environment can induce cellular orientation, a process which may have significant implications for the formation and mechanical properties of tissues.

  5. Subcellular Size

    PubMed Central

    Marshall, Wallace F.

    2016-01-01

    All of the same conceptual questions about size in organisms apply equally at the level of single cells. What determines the size, not only of the whole cell, but of all its parts? What ensures that subcellular components are properly proportioned relative to the whole cell? How does alteration in organelle size affect biochemical function? Answering such fundamental questions requires us to understand how the size of individual organelles and other cellular structures is determined. Knowledge of organelle biogenesis and dynamics has advanced rapidly in recent years. Does this knowledge give us enough information to formulate reasonable models for organelle size control, or are we still missing something? PMID:25957302

  6. [X-ray irradiation induces apoptosis of mouse GC1 sperm cells via nuclear translocation of apoptosis-inducing factor].

    PubMed

    Yang, Huiying; Ding, Jingbin; Wang, Zhijun; Ding, Juan; Xia, Xinshe; Zhao, Wei

    2017-03-01

    Objective To study the effect of X-ray irradiation on the localization of apoptosis inducing factor (AIF) in mouse GC1 sperm cells. Methods After GC1 cells were treated with 0, 3, 6 and 9 Gy X irradiation, BrdU incorporation assay was performed to detect the proliferation of GC1 cells. Forty-eight hours after irradiation, the nuclear condensation was observed by DAPI staining. The subcellular localization of AIF was showed using the immunofluorescence staining, both in the whole cell extracts and in nuclear extracts, and the expression levels of AIF were detected using Western blot analysis. Results With the increase of X-ray irradiation dose, the proliferation of GC1 cells significantly decreased, and the activity of cells was weakened. After 6 Gy irradiation, in nuclear extracts, but not in the whole cell extracts, the protein AIF was upregulated significantly. It meant the nuclear translocation of protein AIF. Conclusion X-ray irradiation induces the apoptosis of mouse GC1 sperm cells, meanwhile, the nuclear translocation of AIF occurs.

  7. Subcellular real-time in vivo imaging of intralymphatic and intravascular cancer-cell trafficking

    NASA Astrophysics Data System (ADS)

    McElroy, M.; Hayashi, K.; Kaushal, S.; Bouvet, M.; Hoffman, Robert M.

    2008-02-01

    With the use of fluorescent cells labeled with green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm and a highly sensitive small animal imaging system with both macro-optics and micro-optics, we have developed subcellular real-time imaging of cancer cell trafficking in live mice. Dual-color cancer cells were injected by a vascular route in an abdominal skin flap in nude mice. The mice were imaged with an Olympus OV100 small animal imaging system with a sensitive CCD camera and four objective lenses, parcentered and parfocal, enabling imaging from macrocellular to subcellular. We observed the nuclear and cytoplasmic behavior of cancer cells in real time in blood vessels as they moved by various means or adhered to the vessel surface in the abdominal skin flap. During extravasation, real-time dual-color imaging showed that cytoplasmic processes of the cancer cells exited the vessels first, with nuclei following along the cytoplasmic projections. Both cytoplasm and nuclei underwent deformation during extravasation. Different cancer cell lines seemed to strongly vary in their ability to extravasate. We have also developed real-time imaging of cancer cell trafficking in lymphatic vessels. Cancer cells labeled with GFP and/or RFP were injected into the inguinal lymph node of nude mice. The labeled cancer cells trafficked through lymphatic vessels where they were imaged via a skin flap in real-time at the cellular level until they entered the axillary lymph node. The bright dual-color fluorescence of the cancer cells and the real-time microscopic imaging capability of the Olympus OV100 enabled imaging the trafficking cancer cells in both blood vessels and lymphatics. With the dual-color cancer cells and the highly sensitive imaging system described here, the subcellular dynamics of cancer metastasis can now be observed in live mice in real time.

  8. ICSI choreography: fate of sperm structures after monospermic rhesus ICSI and first cell cycle implications.

    PubMed

    Ramalho-Santos, J; Sutovsky, P; Simerly, C; Oko, R; Wessel, G M; Hewitson, L; Schatten, G

    2000-12-01

    We have dissected the initial stages of fertilization by intracytoplasmic sperm injection of single spermatozoa into prime oocytes from fertile rhesus monkeys (Macaca mulatta). DNA decondensation was delayed at the apical portion of the sperm head. It is possible that this asynchronous male DNA decondensation could be related to the persistence of the sperm acrosome and perinuclear theca after injection. However, incomplete male pronuclear formation did not prevent sperm aster formation, microtubule nucleation and pronuclear apposition. In contrast, DNA synthesis was delayed in both pronuclei until the sperm chromatin fully decondensed, indicating that male pronuclear formation constitutes an important checkpoint during the first embryonic cell cycle.

  9. Putative cis-regulatory elements in genes highly expressed in rice sperm cells

    PubMed Central

    2011-01-01

    Background The male germ line in flowering plants is initiated within developing pollen grains via asymmetric division. The smaller cell then becomes totally encased within a much larger vegetative cell, forming a unique "cell within a cell structure". The generative cell subsequently divides to give rise to two non-motile diminutive sperm cells, which take part in double fertilization and lead to the seed set. Sperm cells are difficult to investigate because of their presence within the confines of the larger vegetative cell. However, recently developed techniques for the isolation of rice sperm cells and the fully annotated rice genome sequence have allowed for the characterization of the transcriptional repertoire of sperm cells. Microarray gene expression data has identified a subset of rice genes that show unique or highly preferential expression in sperm cells. This information has led to the identification of cis-regulatory elements (CREs), which are conserved in sperm-expressed genes and are putatively associated with the control of cell-specific expression. Findings We aimed to identify the CREs associated with rice sperm cell-specific gene expression data using in silico prediction tools. We analyzed 1-kb upstream regions of the top 40 sperm cell co-expressed genes for over-represented conserved and novel motifs. Analysis of upstream regions with the SIGNALSCAN program with the PLACE database, MEME and the Mclip tool helped to find combinatorial sets of known transcriptional factor-binding sites along with two novel motifs putatively associated with the co-expression of sperm cell-specific genes. Conclusions Our data shows the occurrence of novel motifs, which are putative CREs and are likely targets of transcriptional factors regulating sperm cell gene expression. These motifs can be used to design the experimental verification of regulatory elements and the identification of transcriptional factors that regulate sperm cell-specific gene expression. PMID

  10. LOCALIZER: subcellular localization prediction of both plant and effector proteins in the plant cell

    PubMed Central

    Sperschneider, Jana; Catanzariti, Ann-Maree; DeBoer, Kathleen; Petre, Benjamin; Gardiner, Donald M.; Singh, Karam B.; Dodds, Peter N.; Taylor, Jennifer M.

    2017-01-01

    Pathogens secrete effector proteins and many operate inside plant cells to enable infection. Some effectors have been found to enter subcellular compartments by mimicking host targeting sequences. Although many computational methods exist to predict plant protein subcellular localization, they perform poorly for effectors. We introduce LOCALIZER for predicting plant and effector protein localization to chloroplasts, mitochondria, and nuclei. LOCALIZER shows greater prediction accuracy for chloroplast and mitochondrial targeting compared to other methods for 652 plant proteins. For 107 eukaryotic effectors, LOCALIZER outperforms other methods and predicts a previously unrecognized chloroplast transit peptide for the ToxA effector, which we show translocates into tobacco chloroplasts. Secretome-wide predictions and confocal microscopy reveal that rust fungi might have evolved multiple effectors that target chloroplasts or nuclei. LOCALIZER is the first method for predicting effector localisation in plants and is a valuable tool for prioritizing effector candidates for functional investigations. LOCALIZER is available at http://localizer.csiro.au/. PMID:28300209

  11. Direct Imaging of Single Cells and Tissue at Subcellular Spatial Resolution Using Transmission Geometry MALDI MS

    PubMed Central

    Zavalin, Andre; Todd, Erik M.; Rawhouser, Patrick D.; Yang, Junhai; Norris, Jeremy L.; Caprioli, Richard M.

    2012-01-01

    The need of cellular and sub-cellular spatial resolution in LDI / MALDI Imaging Mass Spectrometry (IMS) necessitates micron and sub-micron laser spot sizes at biologically relevant sensitivities, introducing significant challenges for MS technology. To this end we have developed a transmission geometry vacuum ion source that allows the laser beam to irradiate the back side of the sample. This arrangement obviates the mechanical / ion optic complications in the source by completely separating the optical lens and ion optic structures. We have experimentally demonstrated the viability of transmission geometry MALDI MS for imaging biological tissues and cells with sub-cellular spatial resolution. Furthermore, we demonstrate that in conjunction with new sample preparation protocols, the sensitivity of this instrument is sufficient to obtain molecular images at sub-micron spatial resolution. PMID:23147833

  12. iTRAQ-based quantitative subcellular proteomic analysis of Avibirnavirus-infected cells.

    PubMed

    Sun, Yanting; Hu, Boli; Fan, Chengfei; Jia, Lu; Zhang, Yina; Du, Aifang; Zheng, Xiaojuan; Zhou, Jiyong

    2015-07-01

    Infectious bursal disease virus (IBDV) enters the host cells via endocytic pathway to achieve viral replication in the cytoplasm. Here, we performed LC-MS/MS coupled with isobaric tags for relative and absolute quantification labeling of differentially abundant proteins of IBDV-infected cells using a subcellular fractionation strategy. We show that the viral infection regulates the abundance and/or subcellular localization of 3211 proteins during early infection. In total, 23 cellular proteins in the cytoplasmic proteome and 34 in the nuclear proteome were significantly altered after virus infection. These differentially abundant proteins are involved in such biological processes as immune response, signal transduction, RNA processing, macromolecular biosynthesis, energy metabolism, virus binding, and cellular apoptosis. Moreover, transcriptional profiles of the 25 genes corresponding to the identified proteins were analyzed by quantitative real-time RT-PCR. Ingenuity Pathway Analysis clustered the differentially abundant proteins primarily into the mTOR pathway, PI3K/Akt pathway, and interferon-β signaling cascades. Confocal microscopy showed colocalization of the viral protein VP3 with host proteins heterogeneous nuclear ribonucleoprotein H1, nuclear factor 45, apoptosis inhibitor 5, nuclear protein localization protein 4 and DEAD-box RNA helicase 42 during the virus infection. Together, these identified subcellular constituents provide important information for understanding host-IBDV interactions and underlying mechanisms of IBDV infection and pathogenesis.

  13. Subcellular Localization of Thiol-Capped CdTe Quantum Dots in Living Cells

    NASA Astrophysics Data System (ADS)

    Zhang, Yu; Mi, Lan; Xiong, Rongling; Wang, Pei-Nan; Chen, Ji-Yao; Yang, Wuli; Wang, Changchun; Peng, Qian

    2009-07-01

    Internalization and dynamic subcellular distribution of thiol-capped CdTe quantum dots (QDs) in living cells were studied by means of laser scanning confocal microscopy. These unfunctionalized QDs were well internalized into human hepatocellular carcinoma and rat basophilic leukemia cells in vitro. Co-localizations of QDs with lysosomes and Golgi complexes were observed, indicating that in addition to the well-known endosome-lysosome endocytosis pathway, the Golgi complex is also a main destination of the endocytosed QDs. The movement of the endocytosed QDs toward the Golgi complex in the perinuclear region of the cell was demonstrated.

  14. Sperm Cell Dynamics in Shallow Chambers

    NASA Astrophysics Data System (ADS)

    Condat, Carlos; Marconi, Veronica; Guidobaldi, Alejandro; Giojalas, Laura; Silhanek, Alejandro; Jeyaram, Yogesh; Moshchalkov, Victor

    2015-03-01

    Self-propelled microorganisms are attracted to surfaces. This makes their dynamic behavior in restricted geometries very different from that observed in the bulk. Here we analyze the motion of spermatozoids confined to shallow chambers, investigating the nature of the cell trajectories and their accumulation near the side boundaries. Observed cell trajectories are composed of a succession of quasi-circular and quasi-linear segments. This suggests that the cells follow a path of intermittent trappings near the top and down surfaces separated by stretches of quasi-free motion near the center of the gap. Use of microstructured petal-shaped edges limits accumulation near the borders and contributes to increase the concentration in the chamber interior. System stabilization occurs over times of the order of minutes, which agrees well with a theoretical estimate that assumes that the cell mean-square displacement is largely due to the quasi-linear segments. Pure quasi-circular trajectories would require several hours to stabilize. Our estimates also indicate that stabilization proceeds 2.5 times faster in the rosette geometries than in the smooth-edged chambers, which is another practical reason to prefer the former.

  15. Tissue polarity genes of Drosophila regulate the subcellular location for prehair initiation in pupal wing cells

    PubMed Central

    1993-01-01

    The Drosophila wing is decorated with a regular array of distally pointing hairs. In the pupal wing, the hairs are formed from micro- villus like prehairs that contain large bundles of actin filaments. The distal orientation of the actin bundles reveals the proximal-distal polarity within the pupal wing epithelium. We have used F-actin staining to examine early stages of prehair development in both wild- type and mutant pupal wings. We have found a striking correlation between hair polarity and the subcellular location for assembly of the prehair. In a wild-type wing, all of the distally pointing hairs are derived from prehairs that are formed at the distal vertex of the hexagonally shaped pupal wing cells. Mutations in six tissue polarity genes result in abnormal hair polarity on the adult wing, and all also alter the subcellular location for prehair initiation. Based on their cellular phenotypes, we can place these six genes into three phenotypic groups. Double mutant analysis indicates that these phenotypic groups correspond to epistasis groups. This suggests that the tissue polarity genes function in or on a pathway that controls hair polarity by regulating the subcellular location for prehair formation. PMID:8408199

  16. The deubiquitinating enzyme USP17 is essential for GTPase subcellular localization and cell motility

    PubMed Central

    de la Vega, Michelle; Kelvin, Alyson A.; Dunican, Dara J.; McFarlane, Cheryl; Burrows, James F.; Jaworski, Jakub; Stevenson, Nigel J.; Dib, Karim; Rappoport, Joshua Z.; Scott, Christopher J.; Long, Aideen; Johnston, James A.

    2011-01-01

    Deubiquitinating enzymes are now emerging as potential therapeutic targets that control many cellular processes, but few have been demonstrated to control cell motility. Here, we show that ubiquitin-specific protease 17 (USP17) is rapidly and transiently induced in response to chemokines SDF-1/CXCL12 and IL-8/CXCL8 in both primary cells and cell lines, and that its depletion completely blocks chemokine-induced cell migration and cytoskeletal rearrangements. Using live cell imaging, we demonstrate that USP17 is required for both elongated and amoeboid motility, in addition to chemotaxis. USP17 has previously been reported to disrupt Ras localization and we now find that USP17 depletion blocks chemokine-induced subcellular relocalization of GTPases Cdc42, Rac and RhoA, which are GTPases essential for cell motility. Collectively, these results demonstrate that USP17 has a critical role in cell migration and may be a useful drug target for both inflammatory and metastatic disease. PMID:21448158

  17. Acoustic tweezing cytometry for live-cell subcellular modulation of intracellular cytoskeleton contractility

    PubMed Central

    Fan, Zhenzhen; Sun, Yubing; Di Chen; Tay, Donald; Chen, Weiqiang; Deng, Cheri X.; Fu, Jianping

    2013-01-01

    Mechanical forces are critical to modulate cell spreading, contractility, gene expression, and even stem cell differentiation. Yet, existing tools that can apply controllable subcellular forces to a large number of single cells simultaneously are still limited. Here we report a novel ultrasound tweezing cytometry utilizing ultrasound pulses to actuate functionalized lipid microbubbles covalently attached to single live cells to exert mechanical forces in the pN - nN range. Ultrasonic excitation of microbubbles could elicit a rapid and sustained reactive intracellular cytoskeleton contractile force increase in different adherent mechanosensitive cells. Further, ultrasound-mediated intracellular cytoskeleton contractility enhancement was dose-dependent and required an intact actin cytoskeleton as well as RhoA/ROCK signaling. Our results demonstrated the great potential of ultrasound tweezing cytometry technique using functionalized microbubbles as an actuatable, biocompatible, and multifunctional agent for biomechanical stimulations of cells. PMID:23846290

  18. Survivin promotes oxidative phosphorylation, subcellular mitochondrial repositioning, and tumor cell invasion

    PubMed Central

    Rivadeneira, Dayana B.; Caino, M. Cecilia; Seo, Jae Ho; Angelin, Alessia; Wallace, Douglas C.; Languino, Lucia R.; Altieri, Dario C.

    2015-01-01

    Survivin promotes cell division and suppresses apoptosis in many human cancers, and increased abundance correlates with metastasis and poor prognosis. Here, we showed that a pool of survivin that localized to the mitochondria of certain tumor cell lines enhanced the stability of oxidative phosphorylation Complex II, which promoted cellular respiration. Survivin also supported the subcellular trafficking of mitochondria to the cortical cytoskeleton of tumor cells, which was associated with increased membrane ruffling, increased focal adhesion complex turnover, and increased tumor cell migration and invasion in cultured cells, and enhanced metastatic dissemination in vivo. Therefore, we found that mitochondrial respiration enhanced by survivin contributes to cancer metabolism, and relocalized mitochondria may provide a “regional” energy source to fuel tumor cell invasion and metastasis. PMID:26268608

  19. Isolating Sperm from Cell Mixtures Using Magnetic Beads Coupled with an Anti-PH-20 Antibody for Forensic DNA Analysis

    PubMed Central

    Zhao, Xing-Chun; Wang, Le; Sun, Jing; Jiang, Bo-Wei; Zhang, Er-Li; Ye, Jian

    2016-01-01

    Vaginal swabs taken in rape cases usually contain epithelial cells from the victim and sperm from the assailant and forensic DNA analysis requires separation of sperm from these cell mixtures. PH-20, which is a glycosylphosphatidylinositol-anchored hyaluronidase located on the head of sperm, has important functions in fertilization. Here we describe a newly developed method for sperm isolation using anti-PH-20 antibody-coupled immunomagnetic beads (anti-PH-20 IMBs). Optical microscopy and scanning electron microscopy showed the IMBs recognized the head of sperm specifically and exhibited a great capacity to capture sperm cells. However, we found it necessary to incubate the IMB–sperm complex with DNase I before sperm lysis in order to remove any female DNA completely. We compared the sensitivity of anti-PH-20 IMBs in sperm and epithelial cell discrimination to those coated with a different anti-sperm antibody (anti-SP-10, anti-ADAM2 or anti-JLP). Only the anti-PH-20 IMBs succeeded in isolating sperm from cell mixtures at a sperm/epithelial cell ratio of 103:105. Further, our method exhibited greater power and better stability for sperm isolation compared to the traditional differential lysis strategy. Taken together, the anti-PH-20 IMB method described here could be effective for the isolation of sperm needed to obtain a single-sourced DNA profile as an aid to identifying the perpetrator in sexual assault cases. PMID:27442128

  20. Subcellular control of Rac-GTPase signalling by magnetogenetic manipulation inside living cells

    NASA Astrophysics Data System (ADS)

    Etoc, F.; Lisse, D.; Bellaiche, Y.; Piehler, J.; Coppey, M.; Dahan, M.

    2013-03-01

    Many cell functions rely on the coordinated activity of signalling pathways at a subcellular scale. However, there are few tools capable of probing and perturbing signalling networks with a spatial resolution matching the intracellular dimensions of their activity patterns. Here we present a generic magnetogenetic approach based on the self-assembly of signalling complexes on the surface of functionalized magnetic nanoparticles inside living cells. The nanoparticles act as nanoscopic hot spots that can be displaced by magnetic forces and trigger signal transduction pathways that bring about a cell response. We applied this strategy to Rho-GTPases, a set of molecular switches known to regulate cell morphology via complex spatiotemporal patterns of activity. We demonstrate that the nanoparticle-mediated activation of signalling pathways leads to local remodelling of the actin cytoskeleton and to morphological changes.

  1. Comparative profiling of the sperm proteome.

    PubMed

    Holland, Ashling; Ohlendieck, Kay

    2015-02-01

    The highly complex and species-selective mechanism of fertilization is a central theme of developmental biology. Gametogenesis, sperm activation, and egg-sperm recognition are fundamental biological processes, warranting detailed studies into the molecular composition of gametes. Biological MS has been instrumental for the comprehensive itemizing of gamete proteomes. The protein constellation of sperm cells and its subcellular structures has been established for a variety of animal species. Spermatogenesis and the crucial activation of sperm cells as a prerequisite of successful fertilization and physiological adaptations to external stressors was investigated using proteomics, as well as the underlying mechanisms of male infertility with respect to proteome-wide alterations. This review outlines recent achievements of sperm proteomics and exemplifies the usefulness of gel-based surveys by outlining the comparative analysis of abnormal spermatozoa in globozoospermia. Besides label-free MS techniques and cell-based labeling methodology, high-resolution fluorescence 2DE has been shown to be highly suitable as a proteomic biomarker discovery tool in sperm protein research. The appropriateness of novel protein markers for improving our understanding of normal spermatogenesis and sperm activation versus the molecular pathogenesis of male infertility will be discussed. New biomarker candidates might be useful to improve diagnostic, prognostic, and therapeutic aspects of infertility.

  2. Exogenous DNA internalisation by sperm cells is improved by combining lipofection and restriction enzyme mediated integration.

    PubMed

    Churchil, R R; Gupta, J; Singh, A; Sharma, D

    2011-06-01

    1. Three types of exogenous DNA inserts, i.e. complete linearised pVIVO2-GFP/LacZ vector (9620 bp), the LacZ gene (5317 bp) and the GFP gene (2152 bp) were used to transfect chicken spermatozoa through simple incubation of sperm cells with insert. 2. PCR assay, Dot Blot hybridisation and Southern hybridisation showed the successful internalisation of exogenous DNA by chicken sperm cells. 3. Lipofection and Restriction Enzyme Mediated Integration (REMI) were used to improve the rate of internalisation of exogenous DNA by sperm cells. 4. Results from dot blot as well as Southern hybridisation were semi-quantified and improved exogenous DNA uptake by sperm cells through lipofection and REMI. Stronger signals were observed from hybridisation of LacZ as well as GFP specific probe with the DNA from lipofected exogenous DNA transfected sperm DNA in comparison with those transfected with nude exogenous DNA.

  3. Oxidative DNA Damage to Sperm Cells and Peripheral Blood Leukocytes in Infertile Men

    PubMed Central

    Taken, Kerem; Alp, Hamit Hakan; Eryilmaz, Recep; Donmez, Muhammet Irfan; Demir, Murat; Gunes, Mustafa; Aslan, Rahmi; Sekeroglu, Mehmet Ramazan

    2016-01-01

    Background Oxidative DNA damage is associated with male infertility. The aim of this study was to evaluate the oxidative DNA damage of sperm cells and blood leukocytes and to determine the levels of MDA and NO levels in seminal and blood plasma of idiopathic infertile men. Material/Methods The study enrolled 52 patients, including 30 infertile and 22 fertile men. MDA, NO, and 8-OHdG/106dG were estimated using spectrophotometry and high-pressure liquid chromatography (HPLC)-based methods in seminal and blood plasma. The association with the sperm parameters was assessed, particularly sperm counts and motility. Results The mean sperm concentration and sperm motility of the fertile men were significantly higher than that of the infertile men. The mean MDA and NO concentration in the seminal and blood samples of the infertile men were higher than that of fertile men. Also, the mean numbers of sperm cells and leukocytes 8-OHdG/106dG of the infertile men were significantly higher than that of fertile men (p=0.04 and p<0.001, respectively). Sperm motility and sperm count were negatively correlated with leukocyte and sperm cell 8-OHdG/106dG ratio. However, progressive motility was significantly negatively correlated with sperm cell and leukocyte 8-OHdG/106dG ratio (R=−0.357, p=0.026; R=−0.388, p=0.024, respectively). Conclusions Oxidative stress is an important factor in male infertility. Therefore, biochemical detection of 8-OHdG/106dG in sperm cells and blood leukocytes may be an additional tool in the diagnosis of male infertility. PMID:27837200

  4. Proteomics reveals the importance of the dynamic redistribution of the subcellular location of proteins in breast cancer cells.

    PubMed

    Pinto, Gabriella; Alhaiek, Abdulrab Ahmed M; Godovac-Zimmermann, Jasminka

    2015-02-01

    At the molecular level, living cells are enormously complicated complex adaptive systems in which intertwined genomic, transcriptomic, proteomic and metabolic networks all play a crucial role. At the same time, cells are spatially heterogeneous systems in which subcellular compartmentalization of different functions is ubiquitous and requires efficient cross-compartmental communication. Dynamic redistribution of multitudinous proteins to different subcellular locations in response to cellular functional state is increasingly recognized as a crucial characteristic of cellular function that seems to be at least as important as overall changes in protein abundance. Characterization of the subcellular spatial dynamics of protein distribution is a major challenge for proteomics and recent results with MCF7 breast cancer cells suggest that this may be of particular importance for cancer cells.

  5. An Improved Procedure for Subcellular Spatial Alignment during Live-Cell CLEM

    PubMed Central

    Padman, Benjamin S.; Bach, Markus; Ramm, Georg

    2014-01-01

    Live-cell correlative light and electron microscopy (CLEM) offers unique insights into the ultrastructure of dynamic cellular processes. A critical and technically challenging part of CLEM is the 3-dimensional relocation of the intracellular region of interest during sample processing. We have developed a simple CLEM procedure that uses toner particles from a laser printer as orientation marks. This facilitates easy tracking of a region of interest even by eye throughout the whole procedure. Combined with subcellular fluorescence markers for the plasma membrane and nucleus, the toner particles allow for precise subcellular spatial alignment of the optical and electron microscopy data sets. The toner-based reference grid is printed and transferred onto a polymer film using a standard office printer and laminator. We have also designed a polymer film holder that is compatible with most inverted microscopes, and have validated our strategy by following the ultrastructure of mitochondria that were selectively photo-irradiated during live-cell microscopy. In summary, our inexpensive and robust CLEM procedure simplifies optical imaging, without limiting the choice of optical microscope. PMID:24755651

  6. Global Identification and Differential Distribution Analysis of Glycans in Subcellular Fractions of Bladder Cells

    PubMed Central

    Yang, Ganglong; Huang, Luyu; Zhang, Jiaxu; Yu, Hanjie; Li, Zheng; Guan, Feng

    2016-01-01

    Compartmentalization of cellular components and their associated biological processes is crucial for cellular function. Protein glycosylation provides a basis for diversity of protein functions. Diversity of glycan composition in animal cells remains poorly understood. We used differential centrifugation techniques to isolate four subcellular protein fractions from homogenate of metastatic bladder YTS1 cells, low grade nonmuscle invasive bladder cancer KK47 cells and normal bladder epithelia HCV29 cells: microsomal (Mic), mitochondrial (Mito), nuclear (Nuc), and cytosolic (Cyto). An integrated strategy combining lectin microarray and mass spectrometry (MS) analysis was then applied to evaluate protein glycosylation of the four fractions. Lectin microarray analysis revealed significant differences among the four fractions in terms of glycan binding to the lectins LCA, AAL, MPL, WGA and PWM in YTS1 cell, STL, Jacalin, VVA, LCA and WGA in KK47, and ConA, GNA, VVA and ACA in HCV29 cell. Among a total of 40, 32 and 15 N-glycans in four fractions of three cells detected by MS analysis, high-mannose and fucosylated structures were predominant, 10 N-glycans in YTS1, 5 N-glycans in KK47 and 7 N-glycans in HCV29 were present in all four fractions; and 10 N-glycans in YTS1, 16 N-glycans in KK47, and 3 N-glycans in HCV29 were present in only one fraction. Glycans in the latter category are considered potential markers for the corresponding organelles. The integrated strategy described here allows detailed examination of glycomes subcellular fraction with high resolution and sensitivity, and will be useful for elucidation of the functional roles of glycans and corresponding glycosylated proteins in distinct organelles. PMID:27313494

  7. Comparison of methods for detecting mitomycin C- and ethyl nitrosourea-induced germ cell damage in mice: sperm enzyme activities, sperm motility, and testis weight

    SciTech Connect

    Ficsor, G.; Oldford, G.M.; Loughlin, K.R.; Panda, B.B.; Dubien, J.L.; Ginsberg, L.C.

    1984-01-01

    Testes weights, sperm motility and enzyme activities in single sperm were compared with respect to their ability to detect either developmental or mutational damage to germ cells. Male mice were injected i.p. with 2.5 mg/kg mitomycin C (MC) or 50 or 100 mg/kg ethylnitrosourea (ENU) or saline and were then killed at times such that sperm derived from treated vas sperm (SZ), spermatids (ST), preleptotene-late-spermatogonial cells (PLSG), spermatogonial cells (SG), or spermatogonial stem cells (SGS) could be evaluated. The authors conclude that testis weight, which is easily obtained, is a sensitive indicator of germ cell damage by these agents. Sperm from each animal were evaluated for sperm motility, acrosin activity, succinic dehydrogenase (SDH) activity with or without the competitive inhibitor malonate or after exposure to 60/sup 8/C for 10 min. The latter two assays were to detect sperm enzymes resistant to the inhibitor or heat. The presence of the acrosin protein was also detected immunologically. Of the sperm assays, acrosin activity proved to be the most sensitive indicator of germ cell damage and was the simplest to measure.

  8. In vitro production of haploid sperm cells from male germ cells of foetal cattle.

    PubMed

    Dong, Wu-Zi; Hua, Jin-Lian; Shen, Wen-Zheng; Dou, Zhong-Ying

    2010-04-01

    The purpose of this study was to isolate the foetal cattle male germ cells (mGCs) and then induce them into sperm cells. The mGCs were purified and enriched by a two-step plating method based on the different adherence velocities of mGCs and somatic cells. The percentage of the vasa and the c-kit positive cells were 95.34+/-2.25% and 53.3+/-1.03% by using flow cytometry analysis (FCA), respectively. In feeder-free culture system, the half-suspending cells appeared and formed a 16-cell rosary in medium after the mGCs were cultured for 6-8 days. On immunocytochemical staining during the second passage, some single cells adhering to the plate appeared to be both Oct-4 and alpha6-integrin positive. During the third passage, the mGCs were induced for 48 h by retinol acid (RA) on Sertoli cell-feeder layer, followed by 5-7 days culture in an RA-free medium. Some elongated sperm-like cells appeared in the medium at this stage. We found that the most effective concentration of RA for the inducement was 10(-7)moll(-1) (P<0.01). The haploid cells in suspension were identified by FCA. The elongated sperm-like cells showed proacrosome-like structure and the flagellum with fibre construct under electron microscopy. The mRNA of outer dense fibre-3 (ODF-3) and transcription protein-1 (TP-1) could be detected in the suspended cells by using reverse transcription polymerase chain reaction (RT-PCR). About 23.1% bovine oocytes could be activated to perform cleavage by intracytoplasmic injection with the sperm-like cells, but embryos did not further develop. Our investigation further demonstrated that foetal cattle mGCs could be induced in vitro into haploid sperm in the short term.

  9. Subcellular localization of proteasomes and their regulatory complexes in mammalian cells.

    PubMed Central

    Brooks, P; Fuertes, G; Murray, R Z; Bose, S; Knecht, E; Rechsteiner, M C; Hendil, K B; Tanaka, K; Dyson, J; Rivett, J

    2000-01-01

    Proteasomes can exist in several different molecular forms in mammalian cells. The core 20S proteasome, containing the proteolytic sites, binds regulatory complexes at the ends of its cylindrical structure. Together with two 19S ATPase regulatory complexes it forms the 26S proteasome, which is involved in ubiquitin-dependent proteolysis. The 20S proteasome can also bind 11S regulatory complexes (REG, PA28) which play a role in antigen processing, as do the three variable gamma-interferon-inducible catalytic beta-subunits (e.g. LMP7). In the present study, we have investigated the subcellular distribution of the different forms of proteasomes using subunit specific antibodies. Both 20S proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic and microsomal preparations isolated from rat liver. LMP7 was enriched approximately two-fold compared with core alpha-type proteasome subunits in the microsomal preparations. 20S proteasomes were more abundant than 26S proteasomes, both in liver and cultured cell lines. Interestingly, some significant differences were observed in the distribution of different subunits of the 19S regulatory complexes. S12, and to a lesser extent p45, were found to be relatively enriched in nuclear fractions from rat liver, and immunofluorescent labelling of cultured cells with anti-p45 antibodies showed stronger labelling in the nucleus than in the cytoplasm. The REG was found to be localized predominantly in the cytoplasm. Three- to six-fold increases in the level of REG were observed following gamma-interferon treatment of cultured cells but gamma-interferon had no obvious effect on its subcellular distribution. These results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells and, therefore, that the distribution is more complex than has been reported for yeast proteasomes. PMID:10657252

  10. Subcellular localization of Suppressor of Hairless in Drosophila sense organ cells during Notch signalling.

    PubMed

    Gho, M; Lecourtois, M; Géraud, G; Posakony, J W; Schweisguth, F

    1996-06-01

    During imaginal development of Drosophila, Suppressor of Hairless [Su(H)], an evolutionarily conserved transcription factor that mediates intracellular signalling by the Notch (N) receptor, controls successive alternative cell fate decisions leading to the differentiation of multicellular sensory organs. We describe here the distribution of the Su(H) protein in the wing disc epithelium throughout development of adult sense organs. Su(H) was found to be evenly distributed in the nuclei of all imaginal disc cells during sensory organ precursor cells selection. Thus differential expression and/or subcellular localization of Su(H) is not essential for its function. Soon after division of the pIIa secondary precursor cell, Su(H) specifically accumulates in the nucleus of the future socket cell. At the onset of differentiation of the socket cell, Su(H) is also detected in the cytoplasm. In this differentiating cell, N and deltex participate in the cytoplasmic retention of Su(H). Still, Su(H) does not colocalize with N at the apical-lateral membranes. These observations suggest that N regulates in an indirect manner the cytoplasmic localization of Su(H) in the socket cell. Finally, the pIIb, shaft and socket cells are found to adopt invariant positions along the anteroposterior axis of the notum. This raises the possibility that tissue-polarity biases these N-mediated cell fate choices.

  11. Subcellular targeting and dynamic regulation of PTEN: implications for neuronal cells and neurological disorders

    PubMed Central

    Kreis, Patricia; Leondaritis, George; Lieberam, Ivo; Eickholt, Britta J.

    2014-01-01

    PTEN is a lipid and protein phosphatase that regulates a diverse range of cellular mechanisms. PTEN is mainly present in the cytosol and transiently associates with the plasma membrane to dephosphorylate PI(3,4,5)P3, thereby antagonizing the PI3-Kinase signaling pathway. Recently, PTEN has been shown to associate also with organelles such as the endoplasmic reticulum (ER), the mitochondria, or the nucleus, and to be secreted outside of the cell. In addition, PTEN dynamically localizes to specialized sub-cellular compartments such as the neuronal growth cone or dendritic spines. The diverse localizations of PTEN imply a tight temporal and spatial regulation, orchestrated by mechanisms such as posttranslational modifications, formation of distinct protein–protein interactions, or the activation/recruitment of PTEN downstream of external cues. The regulation of PTEN function is thus not only important at the enzymatic activity level, but is also associated to its spatial distribution. In this review we will summarize (i) recent findings that highlight mechanisms controlling PTEN movement and sub-cellular localization, and (ii) current understanding of how PTEN localization is achieved by mechanisms controlling posttranslational modification, by association with binding partners and by PTEN structural or activity requirements. Finally, we will discuss the possible roles of compartmentalized PTEN in developing and mature neurons in health and disease. PMID:24744697

  12. Expression and subcellular distribution of gephyrin in non-neuronal tissues and cells.

    PubMed

    Nawrotzki, Ralph; Islinger, Markus; Vogel, Ingeborg; Völkl, Alfred; Kirsch, Joachim

    2012-04-01

    Gephyrin is a scaffolding protein required for the accumulation of inhibitory neurotransmitter receptors at neuronal postsynaptic membranes. In non-neuronal tissues, gephyrin is indispensible for the biosynthesis of molybdenum cofactor, the prosthetic group of oxidoreductases including sulfite oxidase and xanthine oxidase. However, the molecular and cellular basis of gephyrin's non-neuronal function is poorly understood; in particular, the roles of its splice variants remain enigmatic. Here, we used cDNA screening as well as Northern and immunoblot analyses to show that mammalian liver contains only a limited number of gephyrin splice variants, with the C3-containing variant being the predominant isoform. Using new and established anti-gephyrin antibodies in immunofluorescence and subcellular fractionation studies, we report that gephyrin localizes to the cytoplasm of both tissue hepatocytes and cultured immortalized cells. These findings were corroborated by RNA interference studies in which the cytosolic distribution was found to be abolished. Finally, by blue-native PAGE we show that cytoplasmic gephyrin is part of a ~600 kDa protein complex of yet unknown composition. Our data suggest that the expression pattern of non-neuronal gephyrin is simpler than indicated by previous evidence. In addition, gephyrin's presence in a cytosolic 600 kDa protein complex suggests that its metabolic and/or other non-neuronal functions are exerted in the cytoplasm and are not confined to a particular subcellular compartment.

  13. Subcellular Localization of GUS- and GFP-Tagged Proteins in Onion Epidermal Cells.

    PubMed

    von Arnim, Albrecht

    2007-02-01

    INTRODUCTIONRecombinant tags (i.e., reporter proteins) offer an excellent alternative to antibodies for determining the subcellular localization of proteins. The most user-friendly tags are the ß-glucuronidase (GUS) reporter enzyme from Escherichia coli and fluorescent proteins derived primarily from the green fluorescent protein (GFP) of the jellyfish Aequorea victoria. GUS is useful primarily as a tag to address nuclear localization, whereas GFP is more versatile. Moreover, GFP is detectable directly in living cells, whereas GUS is only detected indirectly by staining of fixed tissue. This may lead to artifacts or it may obscure problems with protein solubility. In this protocol, protein localization is routinely assayed after particle-mediated transient transformation of onion epidermal cells. With this method it can be determined rapidly whether a given fusion protein is active, and preliminary targeting data can be obtained.

  14. Sex-sorting sperm using flow cytometry/cell sorting.

    PubMed

    Garner, Duane L; Evans, K Michael; Seidel, George E

    2013-01-01

    The sex of mammalian offspring can be predetermined by flow sorting relatively pure living populations of X- and Y-chromosome-bearing sperm. This method is based on precise staining of the DNA of sperm with the nucleic acid-specific fluorophore, Hoechst 33342, to differentiate between the subpopulations of X- and Y-sperm. The fluorescently stained sperm are then sex-sorted using a specialized high speed sorter, MoFlo(®) SX XDP, and collected into biologically supportive media prior to reconcentration and cryopreservation in numbers adequate for use with artificial insemination for some species or for in vitro fertilization. Sperm sorting can provide subpopulations of X- or Y-bearing bovine sperm at rates in the 8,000 sperm/s range while maintaining; a purity of 90% such that it has been applied to cattle on a commercial basis. The sex of offspring has been predetermined in a wide variety of mammalian species including cattle, swine, horses, sheep, goats, dogs, cats, deer, elk, dolphins, water buffalo as well as in humans using flow cytometric sorting of X- and Y-sperm.

  15. Subcellular localisation of VEGF in different pituitary cells. Changes of its expression in oestrogen induced prolactinomas.

    PubMed

    Mukdsi, Jorge Humberto; De Paul, Ana Lucía; Gutiérrez, Silvina; Roth, Félix Daniel; Aoki, Agustín; Torres, Alicia Inés

    2005-10-01

    Vascular endothelial growth factor (VEGF) is an important angiogenic factor in the pituitary gland. The objective of this study was to unveil the VEGF subcellular localisation in different pituitary cell types and to evaluate changes in its expression at different time intervals after oestrogen stimulation. A relevant feature demonstrated was the identification of this cytokine in the nucleus and cytoplasm of lactotrophs, somatotrophs and gonadotrophs, as well as in follicle-stellate cells of male rats. Oestrogen treatment increased the number of VEGF immunopositive cells and its expression detected differentially by western blot in both nucleus and cytoplasm of pituitary cells when compared to the control. At ultrastructural level VEGF appeared associated with nucleolus and euchromatin involving a possible internal autocrine loop. In lactotrophs, the predominant cell of the tumour, VEGF was immunodetected in RER, Golgi complex, and vesicular organelles, supporting further the association with an auto-paracrine effect exerted by VEGF. The nucleus/cytoplasm ratio of VEGF revealed a prevalent accumulation of VEGF in the cytoplasm. The presence of VEGF in the nucleus may probably be associated with a translocation to this cell compartment. This study demonstrated a cytoplasmic and nuclear immunolocalisation of VEGF in normal and tumoural adenohypophyseal cells. In the course of prolactinoma development, the oestrogen stimulated VEGF expression in tumoural cells, promoting a vascular adaptation which contributes to growth and progression of the tumour.

  16. Nanoimaging granule dynamics and subcellular structures in activated mast cells using soft X-ray tomography

    PubMed Central

    Chen, Huan-Yuan; Chiang, Dapi Meng-Lin; Lin, Zi-Jing; Hsieh, Chia-Chun; Yin, Gung-Chian; Weng, I.-Chun; Guttermann, Peter; Werner, Stephan; Henzler, Katja; Schneider, Gerd; Lai, Lee-Jene; Liu, Fu-Tong

    2016-01-01

    Mast cells play an important role in allergic responses. During activation, these cells undergo degranulation, a process by which various kinds of mediators stored in the granules are released. Granule homeostasis in mast cells has mainly been studied by electron microscopy (EM), where the fine structures of subcellular organelles are partially destroyed during sample preparation. Migration and fusion of granules have not been studied in detail in three dimensions (3D) in unmodified samples. Here, we utilized soft X-ray tomography (SXT) coupled with fluorescence microscopy to study the detailed structures of organelles during mast cell activation. We observed granule fission, granule fusion to plasma membranes, and small vesicles budding from granules. We also detected lipid droplets, which became larger and more numerous as mast cells were activated. We observed dramatic morphological changes of mitochondria in activated mast cells and 3D-reconstruction revealed the highly folded cristae inner membrane, features of functionally active mitochondria. We also observed giant vesicles containing granules, mitochondria, and lipid droplets, which we designated as granule-containing vesicles (GCVs) and verified their presence by EM in samples prepared by cryo-substitution, albeit with a less clear morphology. Thus, our studies using SXT provide significant insights into mast cell activation at the organelle level. PMID:27748356

  17. Subcellular and supracellular mechanical stress prescribes cytoskeleton behavior in Arabidopsis cotyledon pavement cells

    PubMed Central

    Sampathkumar, Arun; Krupinski, Pawel; Wightman, Raymond; Milani, Pascale; Berquand, Alexandre; Boudaoud, Arezki; Hamant, Olivier; Jönsson, Henrik; Meyerowitz, Elliot M

    2014-01-01

    Although it is a central question in biology, how cell shape controls intracellular dynamics largely remains an open question. Here, we show that the shape of Arabidopsis pavement cells creates a stress pattern that controls microtubule orientation, which then guides cell wall reinforcement. Live-imaging, combined with modeling of cell mechanics, shows that microtubules align along the maximal tensile stress direction within the cells, and atomic force microscopy demonstrates that this leads to reinforcement of the cell wall parallel to the microtubules. This feedback loop is regulated: cell-shape derived stresses could be overridden by imposed tissue level stresses, showing how competition between subcellular and supracellular cues control microtubule behavior. Furthermore, at the microtubule level, we identified an amplification mechanism in which mechanical stress promotes the microtubule response to stress by increasing severing activity. These multiscale feedbacks likely contribute to the robustness of microtubule behavior in plant epidermis. DOI: http://dx.doi.org/10.7554/eLife.01967.001 PMID:24740969

  18. Intercellular communication in Arabidopsis thaliana pollen discovered via AHG3 transcript movement from the vegetative cell to sperm

    PubMed Central

    Jiang, Hua; Yi, Jun; Boavida, Leonor C.; Chen, Yuan; Becker, Jörg D.; Köhler, Claudia; McCormick, Sheila

    2015-01-01

    An Arabidopsis pollen grain (male gametophyte) consists of three cells: the vegetative cell, which forms the pollen tube, and two sperm cells enclosed within the vegetative cell. It is still unclear if there is intercellular communication between the vegetative cell and the sperm cells. Here we show that ABA-hypersensitive germination3 (AHG3), encoding a protein phosphatase, is specifically transcribed in the vegetative cell but predominantly translated in sperm cells. We used a series of deletion constructs and promoter exchanges to document transport of AHG3 transcripts from the vegetative cell to sperm and showed that their transport requires sequences in both the 5′ UTR and the coding region. Thus, in addition its known role in transporting sperm during pollen tube growth, the vegetative cell also contributes transcripts to the sperm cells. PMID:26466609

  19. Identification of Proteins Enriched in Rice Egg or Sperm Cells by Single-Cell Proteomics

    PubMed Central

    Abiko, Mafumi; Furuta, Kensyo; Yamauchi, Yoshio; Fujita, Chiharu; Taoka, Masato; Isobe, Toshiaki; Okamoto, Takashi

    2013-01-01

    In angiosperms, female gamete differentiation, fertilization, and subsequent zygotic development occur in embryo sacs deeply embedded in the ovaries. Despite their importance in plant reproduction and development, how the egg cell is specialized, fuses with the sperm cell, and converts into an active zygote for early embryogenesis remains unclear. This lack of knowledge is partly attributable to the difficulty of direct analyses of gametes in angiosperms. In the present study, proteins from egg and sperm cells obtained from rice flowers were separated by one-dimensional polyacrylamide gel electrophoresis and globally identified by highly sensitive liquid chromatography coupled with tandem mass spectroscopy. Proteome analyses were also conducted for seedlings, callus, and pollen grains to compare their protein expression profiles to those of gametes. The proteomics data have been deposited to the ProteomeXchange with identifier PXD000265. A total of 2,138 and 2,179 expressed proteins were detected in egg and sperm cells, respectively, and 102 and 77 proteins were identified as preferentially expressed in egg and sperm cells, respectively. Moreover, several rice or Arabidopsis lines with mutations in genes encoding the putative gamete-enriched proteins showed clear phenotypic defects in seed set or seed development. These results suggested that the proteomic data presented in this study are foundational information toward understanding the mechanisms of reproduction and early development in angiosperms. PMID:23936051

  20. Identification of proteins enriched in rice egg or sperm cells by single-cell proteomics.

    PubMed

    Abiko, Mafumi; Furuta, Kensyo; Yamauchi, Yoshio; Fujita, Chiharu; Taoka, Masato; Isobe, Toshiaki; Okamoto, Takashi

    2013-01-01

    In angiosperms, female gamete differentiation, fertilization, and subsequent zygotic development occur in embryo sacs deeply embedded in the ovaries. Despite their importance in plant reproduction and development, how the egg cell is specialized, fuses with the sperm cell, and converts into an active zygote for early embryogenesis remains unclear. This lack of knowledge is partly attributable to the difficulty of direct analyses of gametes in angiosperms. In the present study, proteins from egg and sperm cells obtained from rice flowers were separated by one-dimensional polyacrylamide gel electrophoresis and globally identified by highly sensitive liquid chromatography coupled with tandem mass spectroscopy. Proteome analyses were also conducted for seedlings, callus, and pollen grains to compare their protein expression profiles to those of gametes. The proteomics data have been deposited to the ProteomeXchange with identifier PXD000265. A total of 2,138 and 2,179 expressed proteins were detected in egg and sperm cells, respectively, and 102 and 77 proteins were identified as preferentially expressed in egg and sperm cells, respectively. Moreover, several rice or Arabidopsis lines with mutations in genes encoding the putative gamete-enriched proteins showed clear phenotypic defects in seed set or seed development. These results suggested that the proteomic data presented in this study are foundational information toward understanding the mechanisms of reproduction and early development in angiosperms.

  1. Alkali-labile sites in sperm cells from Sus and Ovis species.

    PubMed

    Cortés-Gutiérrez, Elva I; Dávila-Rodríguez, Martha I; López-Fernández, Carmen; Fernández, José Luis; Gosálvez, Jaime

    2008-06-01

    Constitutive alkali-labile sites (ALSs) have been investigated using a protocol of DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) in sperm cells from Sus domesticus (pig), Ovis gmelini musimon (mouflon) and Ovis aries (sheep). The results were compared with those obtained using leucocytes from the same species. Whole comparative genomic hybridization (W-CGH) showed that most of the constitutive ALSs in somatic and germ line cells in all species examined were constrained to particular repetitive satellite DNA sequences located in the pericentromeric constitutive heterochromatin of each chromosome. However, their relative abundance was different among cells of the same organism (leucocytes/sperm cells), and this trend was not maintained when the different species were compared. Thus, in mouflon, the density of ALSs in leucocytes when compared with that observed in sperm cells indicated abundance of the order of eight times less. In sheep, both leucocytes and sperm cells exhibited a large quantity of ALSs, being of the order of four times more abundant in sperm cells. In the pig genome, leucocytes showed a high abundance of ALSs (of the order of 12 times more that in sperm cells) but only involved the metacentric chromosomes of the karyotype. ALSs were not present in the acrocentric chromosomes. Contrary to mouflon and sheep, ALSs were relatively scarce in sperm cells from pig. These results suggest that ALSs are a transient structural feature in the cells of any organisms and point to a non-universal model of chromatin organization in sperm cells among mammals.

  2. Immature dendritic cell-derived exosomes: a promise subcellular vaccine for autoimmunity.

    PubMed

    Yin, Weifan; Ouyang, Song; Li, Yi; Xiao, Bo; Yang, Huan

    2013-02-01

    Exosomes, 60-90-nm-sized vesicles, are produced by a large number of cell types, including tumor cells, neurons, astrocytes, hemocytes, intestinal epithelial cells, and so on. Dendritic cell (DC), the most potent professional antigen-presenting cell in the immune system, produces exosomes in the course of maturation. Mature DCs produce exosomes with the ability to elicit potent immunoactivation, resulting in tumor eradication and bacterial or virus elimination. Given the notion that exosomes are stable and easy to be modified artificially, autologous mature DC-derived exosomes have been vaccinated into patients with malignant diseases. In clinical trials utilizing exosomes as therapeutic approaches, researchers observed considerable curative effect with little side effect. However, immature or suppressive DC-derived exosomes harbor anti-inflammatory properties distinct from mature DC-derived exosomes. In murine models of autoimmune disease and transplantation, immature DC-derived exosomes reduced T cell-dependent immunoactivation, relieved clinical manifestation of autoimmune disease, and prolonged survival time of transplantation. Although the exact mechanism of how immature DC-derived exosomes function in vivo is still unclear, and there are no clinical trials regarding application of exosome vaccine into patients with autoimmune disease, we will analyze the promise of immature DC-derived exosomes as a subcellular vaccine in autoimmunity in this review.

  3. Subcellular localization of human heparanase and its alternative splice variant in COS-7 cells.

    PubMed

    Sato, Mayumi; Amemiya, Kana; Hayakawa, Sumio; Munakata, Hiroshi

    2008-08-01

    Heparanase, the enzyme that degrades heparan sulfate, has been implicated to play important and characteristic roles in organogenesis, tissue organization, cell migration, and tumor metastasis. Clarification of its expression, its intracellular sorting, and its secretion is, therefore, of much importance to understand its role in cell biology. In addition to the 1.7 Kb transcript previously reported, we detected a 1.5 Kb transcript of human heparanase by RT-PCR. The smaller transcript was shown to be an alternatively spliced variant lacking exon 5, which contains the essential glutamic acid residue required for enzyme activity. When expressed in COS-7 cells this variant did not show any heparanase activity. Full-length heparanase and the exon 5-deleted splice variant were expressed in COS-7 cells and examined by confocal laser scanning microscopy. Both proteins co-localized with calnexin, a marker protein for the endoplasmic reticulum, and they co-immunoprecipitated with calnexin. Both proteins were postulated to be precursors based upon the results of SDS-PAGE analyses. Treatment with endoglycosidases revealed that all potential N-glycosylation sites in the proteins were glycosylated. Tunicamycin treatment of transfected COS-7 cells inhibited N-glycosylation but did not change the subcellular localization. These results indicate that overexpressed heparanase and its splice variant localize to the endoplasmic reticulum independent of glycosylation in COS-7 cells.

  4. Phagocytosis of sperm by follicle cells of the carnivorous sponge Asbestopluma occidentalis (Porifera, Demospongiae).

    PubMed

    Riesgo, Ana

    2010-06-01

    During spermatogenesis of the carnivorous sponge Asbestopluma occidentalis, follicle cells that lined the spermatocysts phagocytosed unreleased mature sperm. Such follicle cells are part of the complex envelope that limits spermatocysts of A. occidentalis, which is also comprised of a collagen layer, a thick layer of intertwined cells, and spicules. Follicle cells showed vesicles containing single phagocytosed spermatozoa within their cytoplasm. Additionally, lipids and other inclusions were observed within the cytoplasm of follicle cells. It is likely that follicle cells recapture nutrients by phagocytosing spermatozoa and use them to form lipids and other inclusions. Such sperm phagocytosis is usually performed in higher invertebrates and vertebrates by Sertoli cells that are located in the testis wall. While Sertoli cells develop a wide range of functions such as creating a blood-testis barrier, providing crucial factors to ensure correct progression of spermatogenesis, and phagocytosis of aberrant, degenerating, and unreleased sperm cells, sponge follicle cells may only display phagocytotic activity on spermatogenic cells.

  5. Neonatal anoxia in rats: hippocampal cellular and subcellular changes related to cell death and spatial memory.

    PubMed

    Takada, S H; dos Santos Haemmerle, C A; Motta-Teixeira, L C; Machado-Nils, A V; Lee, V Y; Takase, L F; Cruz-Rizzolo, R J; Kihara, A H; Xavier, G F; Watanabe, I-S; Nogueira, M I

    2015-01-22

    Neonatal anoxia in rodents has been used to understand brain changes and cognitive dysfunction following asphyxia. This study investigated the time-course of cellular and subcellular changes and hippocampal cell death in a non-invasive model of anoxia in neonatal rats, using Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL) to reveal DNA fragmentation, Fluoro-Jade® B (FJB) to show degenerating neurons, cleaved caspase-3 immunohistochemistry (IHC) to detect cells undergoing apoptosis, and transmission electron microscopy (TEM) to reveal fine ultrastructural changes related to cell death. Anoxia was induced by exposing postnatal day 1 (P1) pups to a flow of 100% gaseous nitrogen for 25 min in a chamber maintained at 37 °C. Control rats were similarly exposed to this chamber but with air flow instead of nitrogen. Brain changes following anoxia were evaluated at postnatal days 2, 14, 21 and 60 (P2, P14, P21 and P60). In addition, spatial reference memory following anoxia and control treatments was evaluated in the Morris water maze, starting at P60. Compared to their respective controls, P2 anoxic rats exhibited (1) higher TUNEL labeling in cornus ammonis (CA) 1 and the dentate gyrus (DG), (2) higher FJB-positive cells in the CA2-3, and (3) somato-dendritic swelling, mitochondrial injury and chromatin condensation in irregular bodies, as well as other subcellular features indicating apoptosis, necrosis, autophagy and excitotoxicity in the CA1, CA2-3 and DG, as revealed by TEM. At P14, P21 and P60, both groups showed small numbers of TUNEL-positive and FJB-positive cells. Stereological analysis at P2, P14, P21 and P60 revealed a lack of significant differences in cleaved caspase-3 IHC between anoxic and control subjects. These results suggest that the type of hippocampal cell death following neonatal anoxia is likely independent of caspase-3 activation. Neonatal anoxia induced deficits in acquisition and performance of spatial reference

  6. Intercellular communication in Arabidopsis thaliana pollen discovered via AHG3 transcript movement from the vegetative cell to sperm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An Arabidopsis pollen grain (male gametophyte) consists of three cells: the vegetative cell, which forms the pollen tube, and two sperm cells enclosed within the vegetative cell. It is still unclear if there is intercellular communication between the vegetative cell and the sperm cells. Here we show...

  7. Label-free biochemical characterization of bovine sperm cells using Raman microscopy

    NASA Astrophysics Data System (ADS)

    De Luca, A. C.; Managò, S.; Ferrara, M. A.; Sirleto, L.; Puglisi, R.; Balduzzi, D.; Galli, A.; Rendina, I.; Ferraro, P.; Coppola, G.

    2014-02-01

    The current study relates to a Raman spectroscopy-based method for addressing the problem of sex assessment in mammals. A direct method for sex predetermination in animals is based on the X- and Y-bearing sperm cells sorting before insemination. Our Raman spectroscope allows distinguishing and characterizing the difference between X- and Y-bearing sperm cells by detecting and analyzing their Raman spectra in a non-invasive and non-destructive way.

  8. Cellular uptake, subcellular distribution and toxicity of arsenic compounds in methylating and non-methylating cells.

    PubMed

    Dopp, E; von Recklinghausen, U; Diaz-Bone, R; Hirner, A V; Rettenmeier, A W

    2010-07-01

    Arsenic is a known human carcinogen, inducing tumors of the skin, urinary bladder, liver and lung. Inorganic arsenic, existing in highly toxic trivalent and significantly less toxic pentavalent forms, is methylated to mono- and di-methylated species mainly in the liver. Due to the low toxicity of pentavalent methylated species, methylation has been regarded as a detoxification process for many years; however, recent findings of a high toxicity of trivalent methylated species have indicated the contrary. In order to elucidate the role of speciation and methylation for the toxicity and carcinogenicity of arsenic, systematic studies were conducted comparing cellular uptake, subcellular distribution as well as toxic and genotoxic effects of organic and inorganic pentavalent and trivalent arsenic species in both non-methylating (urothelial cells and fibroblasts) and methylating cells (hepatocytes). The membrane permeability was found to be dependent upon both the arsenic species and the cell type. Uptake rates of trivalent methylated species were highest and exceeded those of their pentavalent counterparts by several orders of magnitude. Non-methylating cells (urothelial cells and fibroblasts) seem to accumulate higher amounts of arsenic within the cell than the methylating hepatocytes. Cellular uptake and extrusion seem to be faster in hepatocytes than in urothelial cells. The correlation of uptake with toxicity indicates a significant role of membrane permeability towards toxicity. Furthermore, cytotoxic effects are more distinct in hepatocytes. Differential centrifugation studies revealed that elevated concentrations of arsenic are present in the ribosomal fraction of urothelial cells and in nucleic and mitochondrial fractions of hepatic cells. Further studies are needed to define the implications of the observed enrichment of arsenic in specific cellular organelles for its carcinogenic activity. This review summarizes our recent research on cellular uptake

  9. Identification and subcellular localization of molecular complexes of Gq/11α protein in HEK293 cells.

    PubMed

    Drastichova, Zdenka; Novotny, Jiri

    2012-08-01

    Heterotrimeric G-proteins localized in the plasma membrane convey the signals from G-protein-coupled receptors (GPCRs) to different effectors. At least some types of G-protein α subunits have been shown to be partly released from plasma membranes and to move into the cytosol after receptor activation by the agonists. However, the mechanism underlying subcellular redistribution of trimeric G-proteins is not well understood and no definitive conclusions have been reached regarding the translocation of Gα subunits between membranes and cytosol. Here we used subcellular fractionation and clear-native polyacrylamide gel electrophoresis to identify molecular complexes of G(q/11)α protein and to determine their localization in isolated fractions and stability in naïve and thyrotropin-releasing hormone (TRH)-treated HEK293 cells expressing high levels of TRH receptor and G(11)α protein. We identified two high-molecular-weight complexes of 300 and 140 kDa in size comprising the G(q/11) protein, which were found to be membrane-bound. Both of these complexes dissociated after prolonged treatment with TRH. Still other G(q/11)α protein complexes of lower molecular weight were determined in the cytosol. These 70 kDa protein complexes were barely detectable under control conditions but their levels markedly increased after prolonged (4-16 h) hormone treatment. These results support the notion that a portion of G(q/11)α can undergo translocation from the membrane fraction into soluble fraction after a long-term activation of TRH receptor. At the same time, these findings indicate that the redistribution of G(q/11)α is brought about by the dissociation of high-molecular-weight complexes and concomitant formation of low-molecular-weight complexes containing the G(q/11)α protein.

  10. Specific subcellular localization of siRNAs delivered by lipoplex in MCF-7 breast cancer cells.

    PubMed

    Lavigne, Carole; Thierry, Alain R

    2007-10-01

    In order to better understand the mechanism of delivery of siRNAs by lipid-based vectors, we investigated the subcellular distribution of siRNAs directed against cyclin D1 delivered by the DLS system in the breast cancer cell line MCF-7. Cells were treated with cyclopentenone or 17beta-estradiol to modulate the level of expression of cyclin D1 mRNA. We qualitatively observed that siRNA localized to specific cytoplasmic compartments in the periphery of the nucleus in granular-like structures that do not correspond to early endosomal vesicles. In cells treated with either cyclopentenone or 17beta-estradiol cellular distribution of siRNAs was not affected but variations in the amount of siRNAs present in cells were found. We suggest these variations might be associated with the effects of cyclopentenone and 17beta-estradiol in cyclin D1 gene expression. Low cytotoxicity and highly cellular uptake of lipoplexes was observed in the presence of serum indicating that the DLS system could be a useful tool for siRNA vectorization in vitro and in vivo.

  11. Oxidative bioactivation of abacavir in subcellular fractions of human antigen presenting cells.

    PubMed

    Bell, Catherine C; Santoyo Castelazo, Anahi; Yang, Emma L; Maggs, James L; Jenkins, Rosalind E; Tugwood, Jonathan; O'Neill, Paul M; Naisbitt, Dean J; Park, B Kevin

    2013-07-15

    Human exposure to abacavir, a primary alcohol antiretroviral, is associated with the development of immunological drug reactions in individuals carrying the HLA risk allele B*57:01. Interaction of abacavir with antigen presenting cells results in cell activation through an Hsp70-mediated Toll-like receptor pathway and the provision of T-cell antigenic determinants. Abacavir's electrophilic aldehyde metabolites are potential precursors of neoantigens. Herein, we have used mass spectrometry to study the oxidative metabolism of abacavir in EBV-transformed human B-cells. RNA and protein were isolated from the cells and subjected to transcriptomic and mass spectrometric analyses to identify the redox enzymes expressed. Low levels of isomeric abacavir carboxylic acids were detected in subcellular fractions of EBV-transformed human B-cells incubated with abacavir. Metabolite formation was time-dependent but was not reduced by an inhibitor of Class I alcohol dehydrogenases. Relatively high levels of mRNA were detected for several redox enzymes, including alcohol dehydrogenase 5 (Class III), aldehyde dehydrogenases (ALDH3A2, ALDH6A1, and ALDH9A1), CYP1B1, CYP2R1, CYP7B1, and hydroxysteroid dehydrogenase 10. Over 2600 proteins were identified by mass spectrometry. More than 1000 of these proteins exhibited catalytic activity, and 80 were oxido-reductases. This is the first proteomic inventory of enzymes in antigen presenting cells. However, neither of the hepatic alcohol dehydrogenases of Class I which metabolize abacavir in vitro was expressed at the protein level. Nevertheless the metabolic production of abacavir carboxylic acids by B-cell fractions implies abacavir-treated immune cells might be exposed to the drug's protein-reactive aldehyde metabolites in vivo.

  12. Multi-color fluorescence imaging of sub-cellular dynamics of cancer cells in live mice

    NASA Astrophysics Data System (ADS)

    Hoffman, Robert M.

    2006-02-01

    We have genetically engineered dual-color fluorescent cells with one color in the nucleus and the other in the cytoplasm that enables real-time nuclear-cytoplasmic dynamics to be visualized in living cells in the cytoplasm in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) was expressed of the cancer cells, and green fluorescent protein (GFP) linked to histone H2B was expressed in the nucleus. Mitotic cells were visualized by whole-body imaging after injection in the mouse ear. Common carotid artery or heart injection of dual-color cells and a reversible skin flap enabled the external visualization of the dual-color cells in microvessels in the mouse where extreme elongation of the cell body as well as the nucleus occurred. The migration velocities of the dual-color cancer cells in the capillaries were measured by capturing individual images of the dual-color fluorescent cells over time. Human HCT-116-GFP-RFP colon cancer and mouse mammary tumor (MMT)-GFP-RFP cells were injected in the portal vein of nude mice. Extensive clasmocytosis (destruction of the cytoplasm) of the HCT-116-GFP-RFP cells occurred within 6 hours. The data suggest rapid death of HCT-116-GFP-RFP cells in the portal vein. In contrast, MMT-GFP-RFP cells injected into the portal vein mostly survived and formed colonies in the liver. However, when the host mice were pretreated with cyclophosphamide, the HCT-116-GFP-RFP cells also survived and formed colonies in the liver after portal vein injection. These results suggest that a cyclophosphamide-sensitive host cellular system attacked the HCT-116-GFP-RFP cells but could not effectively kill the MMT-GFP-RFP cells. With the ability to continuously image cancer cells at the subcellular level in the live animal, our understanding of the complex steps of metastasis will significantly increase. In addition, new drugs can be developed to target these newly visible steps of metastasis.

  13. Cytoplasmic connection of sperm cells to the pollen vegetative cell nucleus: potential roles of the male germ unit revisited.

    PubMed

    McCue, Andrea D; Cresti, Mauro; Feijó, José A; Slotkin, R Keith

    2011-03-01

    The male germ cells of angiosperm plants are neither free-living nor flagellated and therefore are dependent on the unique structure of the pollen grain for fertilization. During angiosperm male gametogenesis, an asymmetric mitotic division produces the generative cell, which is completely enclosed within the cytoplasm of the larger pollen grain vegetative cell. Mitotic division of the generative cell generates two sperm cells that remain connected by a common extracellular matrix with potential intercellular connections. In addition, one sperm cell has a cytoplasmic projection in contact with the vegetative cell nucleus. The shared extracellular matrix of the two sperm cells and the physical association of one sperm cell to the vegetative cell nucleus forms a linkage of all the genetic material in the pollen grain, termed the male germ unit. Found in species representing both the monocot and eudicot lineages, the cytoplasmic projection is formed by vesicle formation and microtubule elongation shortly after the formation of the generative cell and tethers the male germ unit until just prior to fertilization. The cytoplasmic projection plays a structural role in linking the male germ unit, but potentially plays other important roles. Recently, it has been speculated that the cytoplasmic projection and the male germ unit may facilitate communication between the somatic vegetative cell nucleus and the germinal sperm cells, via RNA and/or protein transport. This review focuses on the nature of the sperm cell cytoplasmic projection and the potential communicative function of the male germ unit.

  14. Subcellular localization of PUMA regulates its pro-apoptotic activity in Burkitt's lymphoma B cells

    PubMed Central

    Ambroise, Gorbatchev; Portier, Alain; Roders, Nathalie; Arnoult, Damien; Vazquez, Aimé

    2015-01-01

    The BH3-only protein PUMA (p53-upregulated modulator of apoptosis) is a major regulator of apoptosis. It belongs to the Bcl-2 family of proteins responsible for maintaining mitochondrial outer membrane integrity by controlling the intrinsic (mitochondrial) apoptotic pathway. We describe here a new pathway regulating PUMA activation through the control of its subcellular distribution. Surprisingly, neither PUMA upregulation in normal activated human B lymphocytes nor high levels of PUMA in Burkitt's lymphoma (BL) were associated with cell death. We show that PUMA is localized to the cytosol in these cells. By contrast, various apoptosis-triggering signals were found to promote the translocation of PUMA to the mitochondria in these cells, leading to their death by apoptosis. This apoptosis was associated with the binding of mitochondrial PUMA to anti-apoptotic members of the Bcl-2 family, such as Bcl-2 and Mcl-1. This translocation was caspase-independent but was prevented by inhibiting or knocking down the expression of the MAPK kinase p38. Our data suggest that the accumulation of PUMA in the cytosol may be important for the participation of this protein in apoptosis without the need for prior transcription. This regulatory pathway may be an important feature of differentiation and tumorigenic processes. PMID:26431330

  15. Subcellular localization of PUMA regulates its pro-apoptotic activity in Burkitt's lymphoma B cells.

    PubMed

    Ambroise, Gorbatchev; Portier, Alain; Roders, Nathalie; Arnoult, Damien; Vazquez, Aimé

    2015-11-10

    The BH3-only protein PUMA (p53-upregulated modulator of apoptosis) is a major regulator of apoptosis. It belongs to the Bcl-2 family of proteins responsible for maintaining mitochondrial outer membrane integrity by controlling the intrinsic (mitochondrial) apoptotic pathway. We describe here a new pathway regulating PUMA activation through the control of its subcellular distribution. Surprisingly, neither PUMA upregulation in normal activated human B lymphocytes nor high levels of PUMA in Burkitt's lymphoma (BL) were associated with cell death. We show that PUMA is localized to the cytosol in these cells. By contrast, various apoptosis-triggering signals were found to promote the translocation of PUMA to the mitochondria in these cells, leading to their death by apoptosis. This apoptosis was associated with the binding of mitochondrial PUMA to anti-apoptotic members of the Bcl-2 family, such as Bcl-2 and Mcl-1. This translocation was caspase-independent but was prevented by inhibiting or knocking down the expression of the MAPK kinase p38. Our data suggest that the accumulation of PUMA in the cytosol may be important for the participation of this protein in apoptosis without the need for prior transcription. This regulatory pathway may be an important feature of differentiation and tumorigenic processes.

  16. Subcellular real-time imaging of the efficacy of temozolomide on cancer cells in the brain of live mice.

    PubMed

    Momiyama, Masashi; Suetsugu, Atsushi; Chishima, Takashi; Bouvet, Michael; Endo, Itaru; Hoffman, Robert M

    2013-01-01

    Novel subcellular imaging technology has been developed in order to visualize drug efficacy on single cancer cells in the brain of mice in real time. The efficacy of temozolomide on cancer cells in the brain was determined by observation of subcellular cancer-cell dynamics over time through a craniotomy open window. Dual-color U87 human glioma and Lewis lung carcinoma (LLC) cells, expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm, were imaged through the craniotomy open window 10 days after treatment with temozolomide (100 mg/kg i.p. for five consecutive days). After treatment, dual-color cancer cells with fragmented nuclei were visualized, indicating apoptosis. GFP-expressing apoptotic bodies and the destruction of RFP-expressing cytoplasm were also visualized. In addition, the terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay was used to confirm apoptosis visualized by imaging of the behavior of GFP-labeled cancer-cell nuclei. Tumor volume in the treated group was significantly smaller than in the control group (at day 19, p<0.001). The present study demonstrates technology capable of subcellular real-time imaging in the brain that reports induction of cancer-cell apoptosis by therapeutic treatment. More effective drugs for brain cancer and brain metastasis can be screened and can be identified with this technology.

  17. Low Concentrations of Hydrogen Peroxide Activate the Antioxidant Defense System in Human Sperm Cells.

    PubMed

    Evdokimov, V V; Barinova, K V; Turovetskii, V B; Muronetz, V I; Schmalhausen, E V

    2015-09-01

    The effect of low concentrations of hydrogen peroxide (10-100 µM) on sperm motility and on the activity of the sperm enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDS) was investigated. Incubation of semen samples with 10 and 100 µM hydrogen peroxide increased the content of spermatozoa with progressive motility by 20 and 18%, respectively, and enhanced the activity of GAPDS in the sperm cells by 27 and 20% compared to a semen sample incubated without additions. It was also found that incubation with 10 µM hydrogen peroxide increased the content of reduced glutathione (GSH) in sperm cells by 50% on average compared to that in the control samples. It is supposed that low concentrations of hydrogen peroxide activate the pentose phosphate pathway, resulting in NADPH synthesis and the reduction of the oxidized glutathione by glutathione reductase yielding GSH. The formed GSH reduces the oxidized cysteine residues of the GAPDS active site, increasing the activity of the enzyme, which in turn enhances the content of sperm cells with progressive motility. Thus, the increase in motile spermatozoa in the presence of low concentrations of hydrogen peroxide can serve as an indicator of normal functioning of the antioxidant defense system in sperm cells.

  18. Effect of bovine oviduct epithelial cell apical plasma membranes on sperm function assessed by a novel flow cytometric approach.

    PubMed

    Boilard, Mathieu; Bailey, Janice; Collin, Simon; Dufour, Maurice; Sirard, Marc-André

    2002-10-01

    In the bovine, as in many mammalian species, sperm are temporarily stored in the oviduct before fertilization by binding to the oviduct epithelial cell apical plasma membranes. As the oviduct is able to maintain motility and viability of sperm and modulate capacitation, we propose that proteins present on the apical plasma membrane of oviduct epithelial cells contribute to these effects. To verify this hypothesis, the motility of frozen-thawed sperm was determined after incubation for 6 h with purified apical plasma membranes from fresh or cultured oviduct epithelial cells or from bovine mammary gland cells as a control. Analysis of intracellular calcium levels was performed by flow cytometry on sperm incubated with fresh membranes using Indo-1 to assess the membrane effect on intracellular calcium concentration. The coculture of sperm with fresh and cultured apical membranes maintained initial motility for 6 h (65% and 84%, respectively). This effect was significantly different from control sperm incubated without oviduct epithelial cell apical membranes (23%), with mammary gland cell apical membranes (23%), or with boiled epithelial cell apical membranes (21%). Apical membranes from oviduct epithelial cells diminished the percentage of sperm that reached a lethal calcium concentration over a 4-h period (18.7%) compared with the control (53.8%) and maintained lower intracellular calcium levels in viable sperm. These results show that the apical plasma membrane of bovine oviduct epithelial cells contains anchored proteinic factors that contribute to maintaining motility and viability and possibly to modulating capacitation of bovine sperm.

  19. Intact Cell MALDI-TOF MS on Sperm: A Molecular Test For Male Fertility Diagnosis.

    PubMed

    Soler, Laura; Labas, Valérie; Thélie, Aurore; Grasseau, Isabelle; Teixeira-Gomes, Ana-Paula; Blesbois, Elisabeth

    2016-06-01

    Currently, evaluation of sperm quality is primarily based on in vitro measures of sperm function such as motility, viability and/or acrosome reaction. However, results are often poorly correlated with fertility, and alternative diagnostic tools are therefore needed both in veterinary and human medicine. In a recent pilot study, we demonstrated that MS profiles from intact chicken sperm using MALDI-TOF profiles could detect significant differences between fertile/subfertile spermatozoa showing that such profiles could be useful for in vitro male fertility testing. In the present study, we performed larger standardized experimental procedures designed for the development of fertility- predictive mathematical models based on sperm cell MALDI-TOF MS profiles acquired through a fast, automated method. This intact cell MALDI-TOF MS-based method showed high diagnostic accuracy in identifying fertile/subfertile males in a large male population of known fertility from two distinct genetic lineages (meat and egg laying lines). We additionally identified 40% of the m/z peaks observed in sperm MS profiles through a top-down high-resolution protein identification analysis. This revealed that the MALDI-TOF MS spectra obtained from intact sperm cells contained a large proportion of protein degradation products, many implicated in important functional pathways in sperm such as energy metabolism, structure and movement. Proteins identified by our predictive model included diverse and important functional classes providing new insights into sperm function as it relates to fertility differences in this experimental system. Thus, in addition to the chicken model system developed here, with the use of appropriate models these methods should effectively translate to other animal taxa where similar tests for fertility are warranted.

  20. Precision Automation of Cell Type Classification and Sub-Cellular Fluorescence Quantification from Laser Scanning Confocal Images

    PubMed Central

    Hall, Hardy C.; Fakhrzadeh, Azadeh; Luengo Hendriks, Cris L.; Fischer, Urs

    2016-01-01

    While novel whole-plant phenotyping technologies have been successfully implemented into functional genomics and breeding programs, the potential of automated phenotyping with cellular resolution is largely unexploited. Laser scanning confocal microscopy has the potential to close this gap by providing spatially highly resolved images containing anatomic as well as chemical information on a subcellular basis. However, in the absence of automated methods, the assessment of the spatial patterns and abundance of fluorescent markers with subcellular resolution is still largely qualitative and time-consuming. Recent advances in image acquisition and analysis, coupled with improvements in microprocessor performance, have brought such automated methods within reach, so that information from thousands of cells per image for hundreds of images may be derived in an experimentally convenient time-frame. Here, we present a MATLAB-based analytical pipeline to (1) segment radial plant organs into individual cells, (2) classify cells into cell type categories based upon Random Forest classification, (3) divide each cell into sub-regions, and (4) quantify fluorescence intensity to a subcellular degree of precision for a separate fluorescence channel. In this research advance, we demonstrate the precision of this analytical process for the relatively complex tissues of Arabidopsis hypocotyls at various stages of development. High speed and robustness make our approach suitable for phenotyping of large collections of stem-like material and other tissue types. PMID:26904081

  1. Patterns of plant subcellular responses to successful oomycete infections reveal differences in host cell reprogramming and endocytic trafficking

    PubMed Central

    Lu, Yi-Ju; Schornack, Sebastian; Spallek, Thomas; Geldner, Niko; Chory, Joanne; Schellmann, Swen; Schumacher, Karin; Kamoun, Sophien; Robatzek, Silke

    2016-01-01

    Summary Adapted filamentous pathogens such as the oomycetes Hyaloperonospora arabidopsidis (Hpa) and Phytophthora infestans (Pi) project specialized hyphae, the haustoria, inside living host cells for the suppression of host defence and acquisition of nutrients. Accommodation of haustoria requires reorganization of the host cell and the biogenesis of a novel host cell membrane, the extrahaustorial membrane (EHM), which envelops the haustorium separating the host cell from the pathogen. Here, we applied live-cell imaging of fluorescent-tagged proteins labelling a variety of membrane compartments and investigated the subcellular changes associated with accommodating oomycete haustoria in Arabidopsis and N. benthamiana. Plasma membrane-resident proteins differentially localized to the EHM. Likewise, secretory vesicles and endosomal compartments surrounded Hpa and Pi haustoria revealing differences between these two oomycetes, and suggesting a role for vesicle trafficking pathways for the pathogen-controlled biogenesis of the EHM. The latter is supported by enhanced susceptibility of mutants in endosome-mediated trafficking regulators. These observations point at host subcellular defences and specialization of the EHM in a pathogen-specific manner. Defence-associated haustorial encasements, a double-layered membrane that grows around mature haustoria, were frequently observed in Hpa interactions. Intriguingly, all tested plant proteins accumulated at Hpa haustorial encasements suggesting the general recruitment of default vesicle trafficking pathways to defend pathogen access. Altogether, our results show common requirements of subcellular changes associated with oomycete biotrophy, and highlight differences between two oomycete pathogens in reprogramming host cell vesicle trafficking for haustoria accommodation. This provides a framework for further dissection of the pathogen-triggered reprogramming of host subcellular changes. PMID:22233428

  2. Patterns of plant subcellular responses to successful oomycete infections reveal differences in host cell reprogramming and endocytic trafficking.

    PubMed

    Lu, Yi-Ju; Schornack, Sebastian; Spallek, Thomas; Geldner, Niko; Chory, Joanne; Schellmann, Swen; Schumacher, Karin; Kamoun, Sophien; Robatzek, Silke

    2012-05-01

    Adapted filamentous pathogens such as the oomycetes Hyaloperonospora arabidopsidis (Hpa) and Phytophthora infestans (Pi) project specialized hyphae, the haustoria, inside living host cells for the suppression of host defence and acquisition of nutrients. Accommodation of haustoria requires reorganization of the host cell and the biogenesis of a novel host cell membrane, the extrahaustorial membrane (EHM), which envelops the haustorium separating the host cell from the pathogen. Here, we applied live-cell imaging of fluorescent-tagged proteins labelling a variety of membrane compartments and investigated the subcellular changes associated with accommodating oomycete haustoria in Arabidopsis and N. benthamiana. Plasma membrane-resident proteins differentially localized to the EHM. Likewise, secretory vesicles and endosomal compartments surrounded Hpa and Pi haustoria revealing differences between these two oomycetes, and suggesting a role for vesicle trafficking pathways for the pathogen-controlled biogenesis of the EHM. The latter is supported by enhanced susceptibility of mutants in endosome-mediated trafficking regulators. These observations point at host subcellular defences and specialization of the EHM in a pathogen-specific manner. Defence-associated haustorial encasements, a double-layered membrane that grows around mature haustoria, were frequently observed in Hpa interactions. Intriguingly, all tested plant proteins accumulated at Hpa haustorial encasements suggesting the general recruitment of default vesicle trafficking pathways to defend pathogen access. Altogether, our results show common requirements of subcellular changes associated with oomycete biotrophy, and highlight differences between two oomycete pathogens in reprogramming host cell vesicle trafficking for haustoria accommodation. This provides a framework for further dissection of the pathogen-triggered reprogramming of host subcellular changes.

  3. Assessing the subcellular distribution of oncogenic phosphoinositide 3-kinase using microinjection into live cells

    PubMed Central

    Layton, Meredith J.; Rynkiewicz, Natalie K.; Ivetac, Ivan; Horan, Kristy A.; Mitchell, Christina A.; Phillips, Wayne A.

    2014-01-01

    Oncogenic mutations in PIK3CA lead to an increase in intrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3Kα (phosphoinositide 3-kinase α) to its PM (plasma membrane) localized substrate is also required for increased levels of downstream PIP3/Akt [phosphoinositide-3,4,5-trisphosphate/also called PKB (protein kinase B)] signalling. We have studied the subcellular localization of wild-type and the two most common oncogenic mutants of PI3Kα in cells maintained in growth media, and starved or stimulated cells using a novel method in which PI3Kα is pre-formed as a 1:1 p110α:p85α complex in vitro then introduced into live cells by microinjection. Oncogenic E545K and H1047R mutants did not constitutively interact with membrane lipids in vitro or in cells maintained in 10% (v/v) FBS. Following stimulation of RTKs (receptor tyrosine kinases), microinjected PI3Kα was recruited to the PM, but oncogenic forms of PI3Kα were not recruited to the PM to a greater extent and did not reside at the PM longer than the wild-type PI3Kα. Instead, the E545K mutant specifically bound activated Cdc42 in vitro and microinjection of E545K was associated with the formation of cellular protrusions, providing some preliminary evidence that changes in protein–protein interactions may play a role in the oncogenicity of the E545K mutant in addition to the well-known changes in lipid kinase activity. PMID:27919038

  4. Separation of sperm and epithelial cells based on the hydrodynamic effect for forensic analysis

    PubMed Central

    Liu, Weiran; Chen, Weixing; Liu, Ran; Ou, Yuan; Liu, Haoran; Xie, Lan; Lu, Ying; Li, Caixia; Li, Bin; Cheng, Jing

    2015-01-01

    In sexual assault cases, forensic samples are a mixture of sperm from the perpetrator and epithelial cells from the victim. To obtain an independent short tandem repeat (STR) profile of the perpetrator, sperm cells must be separated from the mixture of cells. However, the current method used in crime laboratories, namely, differential extraction, is a time-consuming and labor-intensive process. To achieve a rapid and automated sample pretreatment process, we fabricated a microdevice for hydrodynamic and size-based separation of sperm and epithelial cells. When cells in suspension were introduced into the device's microfluidic channels, they were forced to flow along different streamlines and into different outlets due to their different diameters. With the proposed microdevice, sperm can be separated within a short period of time (0.5 h for a 50-μl mock sample). The STR profiles of the products in the sperm outlet reservoir demonstrated that a highly purified male DNA fraction could be obtained (94.0% male fraction). This microdevice is of low-cost and can be easily integrated with other subsequent analysis units, providing great potential in the process of analyzing sexual assault evidence as well as in other areas requiring cell sorting. PMID:26392829

  5. In vitro effect of produced water on cod, Gadus morhua, sperm cells and fertilization.

    PubMed

    Hamoutene, Dounia; Samuelson, S; Lush, L; Burt, K; Drover, D; King, T; Lee, K

    2010-05-01

    The in vitro effect of produced water released by oil and gas platforms was assessed by exposing cod sperm cells to realistic concentrations of this mixture (100, 200, 500 ppm). We investigated produced water impact on enzymes of the aerobic (citrate synthase) and glycolytic metabolism (lactate dehydrogenase), lipid catabolism (lipase), as well as an anti-oxidant enzyme (catalase). Fertilization rates, viability, respiration, ATP, and total motility duration were also evaluated. To explore correlations between these parameters, we have also tested the effect of conserving sperm for 24 h at 4 degrees C. After conservation, fertilization success was decreased but other parameters were not affected. Produced water did not result in a significant change in fertilization; a significant increase in sperm protein amounts and citrate synthase activity can be observed. No correlations are found between parameters showing that sperm viability and unchanged energy levels do not translate into equivalent fertilization capacity. To conclude, exposure of sperm to produced water resulted only in subtle effects on cells. These findings bring information on the effect of produced water on sperm itself rather than on spermatogenesis or testis development of an exposed fish.

  6. Assessment of DNA integrity (COMET assay) in sperm cells of boron-exposed workers.

    PubMed

    Duydu, Yalçin; Başaran, Nurşen; Ustündağ, Aylin; Aydin, Sevtap; Undeğer, Ulkü; Ataman, Osman Yavuz; Aydos, Kaan; Düker, Yalçin; Ickstadt, Katja; Waltrup, Britta Schulze; Golka, Klaus; Bolt, Hermann M

    2012-01-01

    An extension of a male reproductive study conducted in a boric acid/borate production zone at Bandırma, Turkey, is presented. The relation between DNA-strand breaks (COMET assay, neutral and alkaline version) in sperm cells and previously described sperm quality parameters was investigated in boron-exposed males. A correlation between blood boron levels and mean DNA-strand breaks in sperm was weak, and DNA-strand breaks in sperm were statistically not different between control and exposed groups. Therefore, increasing boron exposures had no additional contribution in addition to already pre-existing DNA-strand breaks in the sperm cells. Weak but statistically significant correlations between DNA-strand breaks and motility/morphology parameters of sperm samples were observed in the neutral version of the COMET assay, while correlations between the same variables were statistically not significant in the alkaline version. A likely reason for these negative results, even in highly exposed humans, is that experimental exposures that had led to reproductive toxicity in animals were significantly higher than any boron exposures, which may be reached under realistic human conditions.

  7. The impact of subcellular location on the near infrared-mediated thermal ablation of cells by targeted carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Murali, Vasanth S.; Wang, Ruhung; Mikoryak, Carole A.; Pantano, Paul; Draper, Rockford K.

    2016-10-01

    Single-walled carbon nanotubes (SWNTs) are used in the near infrared (NIR)-mediated thermal ablation of tumor cells because they efficiently convert absorbed NIR light into heat. Despite the therapeutic potential of SWNTs, there have been no published studies that directly quantify how many SWNTs need be associated with a cell to achieve a desired efficiency of killing, or what is the most efficient subcellular location of SWNTs for killing cells. Herein we measured dose response curves for the efficiency of killing correlated to the measured amounts of folate-targeted SWNTs that were either on the surface or within the vacuolar compartment of normal rat kidney cells. Folate-targeted SWNTs on the cell surface were measured after different concentrations of SWNTs in medium were incubated with cells for 30 min at 4 °C. Folate-targeted SWNTs within the vacuolar compartments were measured after cells were incubated with different concentrations of SWNTs in medium for 6 h at 37 °C. It was observed that a SWNT load of ∼13 pg/cell when internalized was sufficient to kill 90% of the cells under standardized conditions of NIR light irradiation. When ∼3.5 pg/cell of SWNTs were internalized within the endosomal/lysosomal compartments, ∼50% of the cells were killed, but when ∼3.5 pg/cell of SWNTs were confined to the cell surface only ∼5% of the cells were killed under the same NIR irradiation conditions. The SWNT subcellular locations were verified using Raman imaging of SWNTs merged with fluorescence images of known subcellular markers. To our knowledge, this is the first time that SWNT amounts at known subcellular locations have been correlated with a dose-normalized efficacy of thermal ablation and the results support the idea that SWNTs confined to the plasma membrane are not as effective in NIR-mediated cell killing as an equivalent amount of SWNTs when internalized within the endosomal/lysosomal vesicles.

  8. Bimodal rheotactic behavior reflects flagellar beat asymmetry in human sperm cells

    PubMed Central

    Bukatin, Anton; Kukhtevich, Igor; Stoop, Norbert; Dunkel, Jörn; Kantsler, Vasily

    2015-01-01

    Rheotaxis, the directed response to fluid velocity gradients, has been shown to facilitate stable upstream swimming of mammalian sperm cells along solid surfaces, suggesting a robust physical mechanism for long-distance navigation during fertilization. However, the dynamics by which a human sperm orients itself relative to an ambient flow is poorly understood. Here, we combine microfluidic experiments with mathematical modeling and 3D flagellar beat reconstruction to quantify the response of individual sperm cells in time-varying flow fields. Single-cell tracking reveals two kinematically distinct swimming states that entail opposite turning behaviors under flow reversal. We constrain an effective 2D model for the turning dynamics through systematic large-scale parameter scans, and find good quantitative agreement with experiments at different shear rates and viscosities. Using a 3D reconstruction algorithm to identify the flagellar beat patterns causing left or right turning, we present comprehensive 3D data demonstrating the rolling dynamics of freely swimming sperm cells around their longitudinal axis. Contrary to current beliefs, this 3D analysis uncovers ambidextrous flagellar waveforms and shows that the cell’s turning direction is not defined by the rolling direction. Instead, the different rheotactic turning behaviors are linked to a broken mirror symmetry in the midpiece section, likely arising from a buckling instability. These results challenge current theoretical models of sperm locomotion. PMID:26655343

  9. Effect of Feeding Fescue Seed Containing Ergot Alkaloid Toxins on Stallion Spermatogenesis and Sperm Cells

    PubMed Central

    Fayrer-Hosken, R; Stanley, A; Hill, N; Heusner, G; Christian, M; Fuente, R De La; Baumann, C; Jones, L

    2012-01-01

    Contents The cellular effects of tall fescue grass–associated toxic ergot alkaloids on stallion sperm and colt testicular tissue were evaluated. This was a continuation of an initial experiment where the effects of toxic ergot alkaloids on the stallion spermiogram were investigated. The only spermiogram parameter in exposed stallions that was affected by the toxic ergot alkaloids was a decreased gel-free volume of the ejaculate. This study examined the effect of toxic ergot alkaloids on chilling and freezing of the stallion sperm cells. The effect of toxic ergot alkaloids on chilled extended sperm cells for 48 h at 5 °C was to make the sperm cells less likely to undergo a calcium ionophore–induced acrosome reaction. The toxic ergot alkaloids had no effect on the freezability of sperm cells. However, if yearling colts were fed toxic ergot alkaloids, then the cytological analysis of meiotic chromosome synapsis revealed a significant increase in the proportion of pachytene spermatocytes showing unpaired sex chromosomes compared to control spermatocytes. There was little effect of ergot alkaloids on adult stallions, but there might be a significant effect on yearling colts. PMID:22524585

  10. Differential Subcellular Localization Renders HAI-2 a Matriptase Inhibitor in Breast Cancer Cells but Not in Mammary Epithelial Cells

    PubMed Central

    Chang, Hsiang-Hua D.; Xu, Yuan; Lai, Hongyu; Yang, Xiaoyu; Tseng, Chun-Che; Lai, Ying-Jung J.; Pan, Yu; Zhou, Emily; Johnson, Michael D.; Wang, Jehng-Kang; Lin, Chen-Yong

    2015-01-01

    The type 2 transmembrane serine protease matriptase is under tight control primarily by the actions of the integral membrane Kunitz-type serine protease inhibitor HAI-1. Growing evidence indicates that HAI-2 might also be involved in matriptase inhibition in some contexts. Here we showed that matriptase inhibition by HAI-2 depends on the subcellular localizations of HAI-2, and is observed in breast cancer cells but not in mammary epithelial cells. HAI-2 is co-expressed with matriptase in 21 out of 26 human epithelial and carcinoma cells examined. HAI-2 is also a potent matriptase inhibitor in solution, but in spite of this, HAI-2 inhibition of matriptase is not observed in all contexts where HAI-2 is expressed, unlike what is seen for HAI-1. Induction of matriptase zymogen activation in mammary epithelial cells results in the formation of matriptase-HAI-1 complexes, but matriptase-HAI-2 complexes are not observed. In breast cancer cells, however, in addition to the appearance of matriptase-HAI-1 complex, three different matriptase-HAI-2 complexes, are formed following the induction of matriptase activation. Immunofluorescent staining reveals that activated matriptase is focused at the cell-cell junctions upon the induction of matriptase zymogen activation in both mammary epithelial cells and breast cancer cells. HAI-2, in contrast, remains localized in vesicle/granule-like structures during matriptase zymogen activation in human mammary epithelial cells. In breast cancer cells, however, a proportion of the HAI-2 reaches the cell surface where it can gain access to and inhibit active matriptase. Collectively, these data suggest that matriptase inhibition by HAI-2 requires the translocation of HAI-2 to the cell surface, a process which is observed in some breast cancer cells but not in mammary epithelial cells. PMID:25786220

  11. Differential subcellular localization renders HAI-2 a matriptase inhibitor in breast cancer cells but not in mammary epithelial cells.

    PubMed

    Chang, Hsiang-Hua D; Xu, Yuan; Lai, Hongyu; Yang, Xiaoyu; Tseng, Chun-Che; Lai, Ying-Jung J; Pan, Yu; Zhou, Emily; Johnson, Michael D; Wang, Jehng-Kang; Lin, Chen-Yong

    2015-01-01

    The type 2 transmembrane serine protease matriptase is under tight control primarily by the actions of the integral membrane Kunitz-type serine protease inhibitor HAI-1. Growing evidence indicates that HAI-2 might also be involved in matriptase inhibition in some contexts. Here we showed that matriptase inhibition by HAI-2 depends on the subcellular localizations of HAI-2, and is observed in breast cancer cells but not in mammary epithelial cells. HAI-2 is co-expressed with matriptase in 21 out of 26 human epithelial and carcinoma cells examined. HAI-2 is also a potent matriptase inhibitor in solution, but in spite of this, HAI-2 inhibition of matriptase is not observed in all contexts where HAI-2 is expressed, unlike what is seen for HAI-1. Induction of matriptase zymogen activation in mammary epithelial cells results in the formation of matriptase-HAI-1 complexes, but matriptase-HAI-2 complexes are not observed. In breast cancer cells, however, in addition to the appearance of matriptase-HAI-1 complex, three different matriptase-HAI-2 complexes, are formed following the induction of matriptase activation. Immunofluorescent staining reveals that activated matriptase is focused at the cell-cell junctions upon the induction of matriptase zymogen activation in both mammary epithelial cells and breast cancer cells. HAI-2, in contrast, remains localized in vesicle/granule-like structures during matriptase zymogen activation in human mammary epithelial cells. In breast cancer cells, however, a proportion of the HAI-2 reaches the cell surface where it can gain access to and inhibit active matriptase. Collectively, these data suggest that matriptase inhibition by HAI-2 requires the translocation of HAI-2 to the cell surface, a process which is observed in some breast cancer cells but not in mammary epithelial cells.

  12. Label-Free Imaging and Biochemical Characterization of Bovine Sperm Cells

    PubMed Central

    Ferrara, Maria Antonietta; Di Caprio, Giuseppe; Managò, Stefano; De Angelis, Annalisa; Sirleto, Luigi; Coppola, Giuseppe; De Luca, Anna Chiara

    2015-01-01

    A full label-free morphological and biochemical characterization is desirable to select spermatozoa during preparation for artificial insemination. In order to study these fundamental parameters, we take advantage of two attractive techniques: digital holography (DH) and Raman spectroscopy (RS). DH presents new opportunities for studying morphological aspect of cells and tissues non-invasively, quantitatively and without the need for staining or tagging, while RS is a very specific technique allowing the biochemical analysis of cellular components with a spatial resolution in the sub-micrometer range. In this paper, morphological and biochemical bovine sperm cell alterations were studied using these techniques. In addition, a complementary DH and RS study was performed to identify X- and Y-chromosome-bearing sperm cells. We demonstrate that the two techniques together are a powerful and highly efficient tool elucidating some important criterions for sperm morphological selection and sex-identification, overcoming many of the limitations associated with existing protocols. PMID:25836358

  13. Morphological evaluation of sperm from infertile men selected by magnetic activated cell sorting (MACS).

    PubMed

    Curti, Gianni; Skowronek, Fernanda; Vernochi, Rita; Rodriguez-Buzzi, Ana Laura; Rodriguez-Buzzi, Juan Carlos; Casanova, Gabriela; Sapiro, Rossana

    2014-12-01

    Electron microscopy analysis performed in five infertile human subjects after sperm selection by swim-up followed by magnetic activated cell sorting (MACS) demonstrated a decrease in the number of spermatozoa with characteristics compatible with cell death. However, no significant differences were found when the swim-up/MACS semen fraction was compared with swim-up fraction alone.

  14. Quantitative evaluation of berberine subcellular distribution and cellular accumulation in non-small cell lung cancer cells by UPLC-MS/MS.

    PubMed

    Yuan, Zhong-Wen; Leung, Elaine Lai-Han; Fan, Xing-Xing; Zhou, Hua; Ma, Wen-Zhe; Liu, Liang; Xie, Ying

    2015-11-01

    Berberine, an isoquinoline alkaloid, has been demonstrated to be a safe anti-cancer agent with multiple effects on mitochondria. Intracellular concentration and distribution around the targeting sites are determinants of efficacy, but subcellular distribution of berberine has not been fully elucidated yet, which relies on the sensitive and robustness assay. In this study, a sensitive and robust UPLC-MS/MS method has been developed and validated with optimized extraction solvents and detection conditions. Key factors such as the purity and integrity of isolated organelle fractions, and the effects of isolation procedures on the subcellular concentration of berberine were systemically evaluated. With the developed assay, we found that the intracellular accumulations of berberine in two gefitinib resistant NSCLC cell lines H1650 and H1975 were 2-3 folds higher than that of normal epithelial cells BEAS-2B. Moreover, significantly different subcellular distribution profiles in NSCLC cancer cells from that of BEAS-2B cells with a striking increase in content in most organelles may contribute to its selective cytotoxicity to cancer cells. Furthermore, a predominant accumulation of berberine was observed for the first time in microsomal fraction for all three cell lines. Therefore, this method could be used for quantitative evaluation of subcellular distribution and cellular accumulation of berberine and for further evaluation of the concentration-effects relationship.

  15. Microthermometry of laser-heated Chinese hamster ovary cells and sperm cells

    NASA Astrophysics Data System (ADS)

    Liu, Yagang; Sonek, Gregory J.; Chapman, Curtis F.; Tromberg, Bruce J.; Patrizio, Pasquale; Tadir, Yona; Berns, Michael W.

    1995-05-01

    Microthermometric measurements on optically-trapped Chinese Hamster Ovary (CHO) cells and sperms cells re reported, using a noninvasive microfluorometric detection technique. Within an optical tweezer system that has been outfitted with a spectral fluorescence excitation and detection capability, the changes in temperature induced by the process of sample confinement by a focused laser beam has been quantified over micron-sized spatial regions of both motile and immotile cells. Our measurement technique is based on the use of environmentally sensitive fluorophores that can be incorporated into the cell membrane and used to sense local changes in temperature when the cell membrane is perturbed optically or via other environmental stress factors. Using a cw 1.064 micrometers Nd:YAG laser for trapping CHO and human sperm cells, a temperature increase of approximately equals 1°C per 100 mW laser power was observed. At this infrared wavelength, cellular heating as result of laser confinement appears to be mainly due to radiation absorption by water.

  16. Divisome-dependent subcellular localization of cell-cell joining protein SepJ in the filamentous cyanobacterium Anabaena.

    PubMed

    Ramos-León, Félix; Mariscal, Vicente; Frías, José E; Flores, Enrique; Herrero, Antonia

    2015-05-01

    Heterocyst-forming cyanobacteria are multicellular organisms that grow as filaments that can be hundreds of cells long. Septal junction complexes, of which SepJ is a possible component, appear to join the cells in the filament. SepJ is a cytoplasmic membrane protein that contains a long predicted periplasmic section and localizes not only to the cell poles in the intercellular septa but also to a position similar to a Z ring when cell division starts suggesting a relation with the divisome. Here, we created a mutant of Anabaena sp. strain PCC 7120 in which the essential divisome gene ftsZ is expressed from a synthetic NtcA-dependent promoter, whose activity depends on the nitrogen source. In the presence of ammonium, low levels of FtsZ were produced, and the subcellular localization of SepJ, which was investigated by immunofluorescence, was impaired. Possible interactions of SepJ with itself and with divisome proteins FtsZ, FtsQ and FtsW were investigated using the bacterial two-hybrid system. We found SepJ self-interaction and a specific interaction with FtsQ, confirmed by co-purification and involving parts of the SepJ and FtsQ periplasmic sections. Therefore, SepJ can form multimers, and in Anabaena, the divisome has a role beyond cell division, localizing a septal protein essential for multicellularity.

  17. Fluorescent berberine binding as a marker of internal glycosaminoglycans sulfate in bovine oocytes and sperm cells.

    PubMed

    Reyes, R; Ramírez, G; Delgado, N M

    2004-01-01

    The use of berberine as a biological marker of glycosamineglycans sulfate was employed to corroborate the presence of heparin in mammalian oocytes and sperm and its distribution in all the structures, or only in some specialized zones, of the male and female gametes. Oocytes and sperms were treated with 1.8 mM berberine for the presence of heparin and examined 10, 30, 60, and 120 minutes later. We have found that heparin is homogeneously distributed in all the zones of bovine oocytes and in sperm cells. When sperm cells are first treated with 80 microM of heparin and then berberine, 40% of them display in their post acrosomal region an intense yellow fluorescence. This may be in relation to the high amount of heparin binding sites due to the presence of the reticular membranous like system in this sperm region and in its possible role whereby gametes recognize and adhere to one another. Therefore, the use of berberine as a fluorescent marker of heparin represents clear proof of the presence of GAGs and their binding sites in the outside and inside of mammalian gametes, reinforcing the importance they play in the events of the process of fertilization.

  18. Effects of X-irradiation on mouse testicular cells and sperm chromatin structure

    SciTech Connect

    Sailer, B.L.; Jost, L.K.; Erickson, K.R.; Tajiran, M.A.; Evenson, D.P.

    1995-07-01

    The testicular regions of male mice were exposed to x-ray doses ranging from 0 to 400 rads. Forty days after exposure the mice were killed and the testes and cauda epididymal sperm removed surgically. Flow cytometric measurements of acridine orange stained testicular samples indicated a repopulation of testicular samples indicated a repopulation of testicular cell types following x-ray killing of stem cells. Cauda epididymal sperm were analyzed by the sperm chromatin structure assay (SCSA), a flow cytometric measurement of the susceptibility of the sperm nuclear DNA to in situ acid denaturation. The SCSA detected increased susceptibility to DNA denaturation in situ after 12.5 rads of x-ray exposure, with significant increases following 25 rads. Abnormal sperm head morphology was not significantly increased until the testes were exposed to 60 rads of x-rays. These data suggest that the SCSA is currently the most sensitive, noninvasive method of detecting x-ray damage to testicular stem spermatogonia. 47 refs., 5 figs.

  19. Choline Dehydrogenase Polymorphism rs12676 Is a Functional Variation and Is Associated with Changes in Human Sperm Cell Function

    PubMed Central

    Johnson, Amy R.; Lao, Sai; Wang, Tongwen; Galanko, Joseph A.; Zeisel, Steven H.

    2012-01-01

    Approximately 15% of couples are affected by infertility and up to half of these cases arise from male factor infertility. Unidentified genetic aberrations such as chromosomal deletions, translocations and single nucleotide polymorphisms (SNPs) may be the underlying cause of many cases of idiopathic male infertility. Deletion of the choline dehydrogenase (Chdh) gene in mice results in decreased male fertility due to diminished sperm motility; sperm from Chdh−/− males have decreased ATP concentrations likely stemming from abnormal sperm mitochondrial morphology and function in these cells. Several SNPs have been identified in the human CHDH gene that may result in altered CHDH enzymatic activity. rs12676 (G233T), a non-synonymous SNP located in the CHDH coding region, is associated with increased susceptibility to dietary choline deficiency and risk of breast cancer. We now report evidence that this SNP is also associated with altered sperm motility patterns and dysmorphic mitochondrial structure in sperm. Sperm produced by men who are GT or TT for rs12676 have 40% and 73% lower ATP concentrations, respectively, in their sperm. rs12676 is associated with decreased CHDH protein in sperm and hepatocytes. A second SNP located in the coding region of IL17BR, rs1025689, is linked to altered sperm motility characteristics and changes in choline metabolite concentrations in sperm. PMID:22558321

  20. Sperm DNA Fragmentation Index and Hyaluronan Binding Ability in Men from Infertile Couples and Men with Testicular Germ Cell Tumor.

    PubMed

    Marchlewska, Katarzyna; Filipiak, Eliza; Walczak-Jedrzejowska, Renata; Oszukowska, Elzbieta; Sobkiewicz, Slawomir; Wojt, Malgorzata; Chmiel, Jacek; Kula, Krzysztof; Slowikowska-Hilczer, Jolanta

    2016-01-01

    Objective. To investigate sperm DNA fragmentation and sperm functional maturity in men from infertile couples (IC) and men with testicular germ cell tumor (TGCT). Materials and Methods. Semen samples were collected from 312 IC men and 23 men with TGCT before unilateral orchiectomy and oncological treatment. The sperm chromatin dispersion test was performed to determine DNA fragmentation index (DFI) and the ability of sperm to bind with hyaluronan (HA) was assessed. Results. In comparison with the IC men, the men with TGCT had a higher percentage of sperm with fragmented DNA (median 28% versus 21%; p < 0.01) and a lower percentage of HA-bound sperm (24% versus 66%; p < 0.001). Normal results of both analyses were observed in 24% of IC men and 4% of men with TGCT. Negative Spearman's correlations were found between DFI and the percentage of HA-bound sperm in the whole group and in IC subjects and those with TGCT analyzed separately. Conclusions. Approximately 76% of IC men and 96% with TGCT awaiting orchiectomy demonstrated DNA fragmentation and/or sperm immaturity. We therefore recommend sperm banking after unilateral orchiectomy, but before irradiation and chemotherapy; the use of such a deposit appears to be a better strategy to obtain functionally efficient sperms.

  1. Sperm DNA Fragmentation Index and Hyaluronan Binding Ability in Men from Infertile Couples and Men with Testicular Germ Cell Tumor

    PubMed Central

    Filipiak, Eliza; Walczak-Jedrzejowska, Renata; Oszukowska, Elzbieta; Sobkiewicz, Slawomir; Wojt, Malgorzata; Chmiel, Jacek; Kula, Krzysztof; Slowikowska-Hilczer, Jolanta

    2016-01-01

    Objective. To investigate sperm DNA fragmentation and sperm functional maturity in men from infertile couples (IC) and men with testicular germ cell tumor (TGCT). Materials and Methods. Semen samples were collected from 312 IC men and 23 men with TGCT before unilateral orchiectomy and oncological treatment. The sperm chromatin dispersion test was performed to determine DNA fragmentation index (DFI) and the ability of sperm to bind with hyaluronan (HA) was assessed. Results. In comparison with the IC men, the men with TGCT had a higher percentage of sperm with fragmented DNA (median 28% versus 21%; p < 0.01) and a lower percentage of HA-bound sperm (24% versus 66%; p < 0.001). Normal results of both analyses were observed in 24% of IC men and 4% of men with TGCT. Negative Spearman's correlations were found between DFI and the percentage of HA-bound sperm in the whole group and in IC subjects and those with TGCT analyzed separately. Conclusions. Approximately 76% of IC men and 96% with TGCT awaiting orchiectomy demonstrated DNA fragmentation and/or sperm immaturity. We therefore recommend sperm banking after unilateral orchiectomy, but before irradiation and chemotherapy; the use of such a deposit appears to be a better strategy to obtain functionally efficient sperms. PMID:27999814

  2. Effect of age on testicular germ cell apoptosis and sperm aneuploidy in MF-1 mice.

    PubMed

    Brinkworth, M H; Schmid, T E

    2003-01-01

    The spontaneous mutation rate in the male germ-line increases with age. The reason for this is unknown, but presumably involves an age-related degeneration in the efficacy of cellular processes. To investigate the possibility that rates of apoptosis and genetic damage (represented by aneuploidy) might vary with age in mice, the testes and sperm of 2- and 12-month-old male MF-1 mice were examined by a modified TUNEL technique and 3-colour sperm-FISH assay, respectively. Sperm were labeled with probes to chromosomes 8, X and Y and 20,000 sperm scored from each of 5 animals per group. A significant increase in gonosomal disomy was found in the aged mice, especially X-X-8. This suggests that advanced paternal age is associated primarily with meiosis II rather than meiosis I disjunction errors. Neither diploidy nor autosomal disomy was affected in the older group. The rate of germ cell apoptosis (apoptotic cells per seminiferous tubule cross-section per animal per group) was higher in the old mice than controls, but not significantly. Considerable inter-animal variability was observed in the older group. The finding of an increase in levels of sperm aneuploidy is novel for 1-year-old mice and confirms the genotoxic effect of ageing in mice. Since apoptosis is assumed to eliminate cells with unrepaired damage, it may be that the apoptotic response in older mice is compromised, resulting in the higher levels of aneuploidy in sperm. However, given the inter-animal variability in testicular germ cell apoptosis, this awaits confirmation.

  3. Improvement of Longevity and Viability of Sperm Cells Isolated from Pollen of Zea mays L. 1

    PubMed Central

    Zhang, Guichang; Williams, Connie M.; Campenot, Mary K.; McGann, Locksley E.; Cass, David D.

    1992-01-01

    Our previous studies showed that the common maize (Zea mays L.) sperm isolation medium (Brewbaker and Kwack salts in 0.44 m sucrose without buffering) caused cell lysis in vitro. In an attempt to remedy this situation, 6 sugars, 10 buffers, 5 pH values, and 3 membrane protective agents were screened to improve longevity and viability of isolated Zea mays sperm cells as estimated by hemacytometry and flow cytometry. Use of 0.55 m galactose in the isolation solution increased sperm yield by 2.5-fold compared with sucrose, and suspension of isolated sperm cells in the galactose solution gave the best longevity among the six sugars. Buffering the galactose solution with 2 mm 2-(N-morpholino)ethanesulfonic acid significantly improved longevity, whereas other buffers had no effect or decreased the longevity and/or viability. Among the five pH values tested (5.0, 6.0, 6.7, 7.0, and 8.0), pH 6.7 appeared to be optimal for maintenance of both longevity and viability. Screening of membrane protectants showed that cysteine caused a rapid decrease in cell viability and increased lysis, whereas dithiothreitol increased the cell numbers but lowered their viability. Addition of 0.1% bovine serum albumin increased cell numbers and viability, and about 70% of the cells remained viable after 72 h of suspension. Cell longevity and viability were also improved in 0.44 m sucrose when the solution was conditioned with 2-(N-morpholino)ethanesulfonic acid and bovine serum albumin. Use of 2-(N-morpholino)ethanesulfonic acid and bovine serum albumin inthe isolation and suspension medium significantly improved the viability and longevity of sperm cells isolated from Zea mays pollen. PMID:16652985

  4. Implications of caveolae in testicular and epididymal myoid cells to sperm motility.

    PubMed

    Oliveira, Regiana L; Parent, Adam; Cyr, Daniel G; Gregory, Mary; Mandato, Craig A; Smith, Charles E; Hermo, Louis

    2016-06-01

    Seminiferous tubules of the testis and epididymal tubules in adult rodents are enveloped by contractile myoid cells, which move sperm and fluids along the male reproductive tract. Myoid cells in the testis influence Sertoli cells by paracrine signaling, but their role in the epididymis is unknown. Electron microscopy revealed that elongated myoid cells formed several concentric layers arranged in a loose configuration. The edges of some myoid cells in a given layer closely approximated one another, and extended small foot-like processes to cells of overlying layers. Gap junction proteins, connexins 32 and 43, were detected within the myoid cell layers by immunohistochemistry. These myoid cells also had caveolae that contained caveolin-1 and cavin-1 (also known as PTRF). The number of caveolae per unit area of plasma membrane was significantly reduced in caveolin-1-deficient mice (Cav1(-/-) ). Morphometric analyses of Cav1-null testes revealed an enlargement in whole-tubule and epithelial profile areas, whereas these parameters were slightly reduced in the epididymis. Although sperm are non-motile as they pass through the proximal epididymis, statistical analyses of cauda epididymidis sperm concentrations revealed no significant differences between wild-type and Cav1(-/-) mice. Motility analyses, however, indicated that sperm velocity parameters were reduced while beat cross frequency was higher in gametes of Cav1(-/-) mice. Thus while caveolae and their associated proteins are not necessary for myoid cell contractility, they appear to be crucial for signaling with the epididymal epithelium to regulate the proper acquisition of sperm motility. Mol. Reprod. Dev. 83: 526-540, 2016. © 2016 Wiley Periodicals, Inc.

  5. Nitric oxide measurements in hTERT-RPE cells and subcellular fractions exposed to low levels of red light

    NASA Astrophysics Data System (ADS)

    Wigle, Jeffrey C.; Castellanos, Cherry C.; Denton, Michael L.; Holwitt, Eric A.

    2014-02-01

    Cells in a tissue culture model for laser eye injury exhibit increased resistance to a lethal pulse of 2.0-μm laser radiation if the cells are first exposed to 2.88 J/cm2 of red light 24 hr prior to the lethal laser exposure. Changes in expression of various genes associated with apoptosis have been observed, but the biochemical link between light absorption and gene expression remains unknown. Cytochome c oxidase (CCOX), in the electron transport chain, is the currentlyhypothesized absorber. Absorption of the red light by CCOX is thought to facilitate displacement of nitric oxide (NO) by O2 in the active site, increasing cellular respiration and intracellular ATP. However, NO is also an important regulator and mediator of numerous physiological processes in a variety of cell and tissue types that is synthesized from l-arginine by NO synthases. In an effort to determine the relative NO contributions from these competing pathways, we measured NO levels in whole cells and subcellular fractions, with and without exposure to red light, using DAF-FM, a fluorescent dye that stoichiometrically reacts with NO. Red light induced a small, but consistently reproducible, increase in fluorescence intensity in whole cells and some subcellular fractions. Whole cells exhibited the highest overall fluorescence intensity followed by (in order) cytosolic proteins, microsomes, then nuclei and mitochondria.

  6. The application of proteomic approaches to the study of mammalian spermatogenesis and sperm function.

    PubMed

    Macleod, Graham; Varmuza, Susannah

    2013-11-01

    Spermatogenesis is the process by which terminally differentiated sperm are produced from male germline stem cells. This complex developmental process requires the coordination of both somatic and germ cells through phases of proliferation, meiosis, and morphological differentiation, to produce the cell responsible for the delivery of the paternal genome. With infertility affecting ~ 15% of all couples, furthering our understanding of spermatogenesis and sperm function is vital for improving the diagnosis and treatment of male factor infertility. The emerging use of proteomic technologies has played an instrumental role in our understanding of spermatogenesis by providing information regarding the genes involved. This article reviews existing proteomic literature regarding spermatogenesis and sperm function, including the proteomic characterization of spermatogenic cell types, subcellular proteomics, post-translational modifications, interactomes, and clinical studies. Future directions in the application of proteomics to the study of spermatogenesis and sperm function are also discussed.

  7. Migration of sperm cells during pollen tube elongation in Arabidopsis thaliana: behavior during transport, maturation and upon dissociation of male germ unit associations.

    PubMed

    Ge, Lili; Gou, Xiaoping; Yuan, Tong; Strout, Greg W; Nakashima, Jin; Blancaflor, Elison B; Tian, Hui Qiao; Russell, Scott D

    2011-02-01

    The promoter sequence of sperm-expressed gene, PzIPT isolated from the S(vn) (sperm associated with the vegetative nucleus) of Plumbago zeylanica, was fused to a green fluorescent protein (GFP) reporter sequence and transformed into Arabidopsis thaliana to better visualize the live behavior of angiosperm sperm cells. Angiosperm sperm cells are not independently motile, migrating in a unique cell-within-a-cell configuration within the pollen tube. Sperm cells occur in association with the vegetative nucleus forming a male germ unit (MGU). In Arabidopsis, GFP was expressed equally in both sperm cells and was observed using a spinning disk confocal microscope, which allowed long duration observation of cells without bleaching or visible laser radiation damage. Pollen activation is reflected by conspicuous movement of sperm and pollen cytoplasm. Upon pollen germination, sperm cells enter the forming tube and become oriented, typically with a sperm cytoplasmic projection leading the sperm cells in the MGU, which remains intact throughout normal pollen tube elongation. Maturational changes, including vacuolization, general rounding and entry into G2, were observed during in vitro culture. When MGUs were experimentally disrupted by mild temperature elevation, sperm cells no longer tracked the growth of the tube and separated from the MGU, providing critical direct evidence that the MGU is a functional unit required for sperm transmission.

  8. In Vitro Effect of Cell Phone Radiation on Motility, DNA Fragmentation and Clusterin Gene Expression in Human Sperm

    PubMed Central

    Zalata, Adel; El-Samanoudy, Ayman Z; Shaalan, Dalia; El-Baiomy, Youssef; Mostafa, Taymour

    2015-01-01

    Background Use of cellular phones emitting radiofrequency electromagnetic field (RF-EMF) has been increased exponentially and become a part of everyday life. This study aimed to investigate the effects of in vitro RF-EMF exposure emitted from cellular phones on sperm motility index, sperm DNA fragmentation and seminal clusterin (CLU) gene expression. Materials and Methods In this prospective study, a total of 124 semen samples were grouped into the following main categories: i. normozoospermia (N, n=26), ii. asthenozoospermia (A, n=32), iii. asthenoteratozoospermia (AT, n=31) and iv. oligoasthenoteratozoospermia (OAT, n=35). The same semen samples were then divided into two portions non-exposed and exposed samples to cell phone radiation for 1 hour. Before and immediately after exposure, both aliquots were subjected to different assessments for sperm motility, acrosin activity, sperm DNA fragmentation and CLU gene expression. Statistical differences were analyzed using paired t student test for comparisons between two sub-groups where p<0.05 was set as significant. Results There was a significant decrease in sperm motility, sperm linear velocity, sperm linearity index, and sperm acrosin activity, whereas there was a significant increase in sperm DNA fragmentation percent, CLU gene expression and CLU protein levels in the exposed semen samples to RF-EMF compared with non-exposed samples in OAT>AT>A>N groups, respectively (p<0.05). Conclusion Cell phone emissions have a negative impact on exposed sperm motility index, sperm acrosin activity, sperm DNA fragmentation and seminal CLU gene expression, especially in OAT cases. PMID:25918601

  9. Association of protein kinase A type I with detergent-resistant structures of mammalian sperm cells.

    PubMed

    Moos, J; Pĕknicová, J; Geussova, G; Philimonenko, V; Hozák, P

    1998-05-01

    The finding that flagellar movement in detergent-permeabilized sperm cells is restored when Mg ATP and cAMP are added implicated detergent-resistant protein kinase A (PKA) in the regulation of sperm motility. It is widely believed that only the PKA regulatory subunit RII can associate with the cytoskeleton and/or organelles. In this paper we used monoclonal antibodies against the PKA catalytic subunit and RI subunit and demonstrated that PKA type I is also associated with the sperm cytoskeleton. To our knowledge, this is the first report showing anchored PKA type I. This association was found in sperm of nonrodent mammalian species and, to a lesser extent, also in mouse sperm. The PKA catalytic subunit is bound to the cytoskeleton secondarily via its complex with the regulatory subunit. The detergent-resistant complexes of RI and catalytic subunits localize predominantly to the flagellum. Ultrastructural immunogold labeling revealed the association of detergent-resistant PKA type I with outer dense fibers (ODF) and the fibrous sheath (FS) but not with microtubules. This location is consistent with a proposed role of PKA in regulation of FS sliding on underlying ODF.

  10. Displacement correlations between a single mesenchymal-like cell and its nucleus effectively link subcellular activities and motility in cell migration analysis

    PubMed Central

    Lan, Tian; Cheng, Kai; Ren, Tina; Arce, Stephen Hugo; Tseng, Yiider

    2016-01-01

    Cell migration is an essential process in organism development and physiological maintenance. Although current methods permit accurate comparisons of the effects of molecular manipulations and drug applications on cell motility, effects of alterations in subcellular activities on motility cannot be fully elucidated from those methods. Here, we develop a strategy termed cell-nuclear (CN) correlation to parameterize represented dynamic subcellular activities and to quantify their contributions in mesenchymal-like migration. Based on the biophysical meaning of the CN correlation, we propose a cell migration potential index (CMPI) to measure cell motility. When the effectiveness of CMPI was evaluated with respect to one of the most popular cell migration analysis methods, Persistent Random Walk, we found that the cell motility estimates among six cell lines used in this study were highly consistent between these two approaches. Further evaluations indicated that CMPI can be determined using a shorter time period and smaller cell sample size, and it possesses excellent reliability and applicability, even in the presence of a wide range of noise, as might be generated from individual imaging acquisition systems. The novel approach outlined here introduces a robust strategy through an analysis of subcellular locomotion activities for single cell migration assessment. PMID:27670131

  11. Displacement correlations between a single mesenchymal-like cell and its nucleus effectively link subcellular activities and motility in cell migration analysis

    NASA Astrophysics Data System (ADS)

    Lan, Tian; Cheng, Kai; Ren, Tina; Arce, Stephen Hugo; Tseng, Yiider

    2016-09-01

    Cell migration is an essential process in organism development and physiological maintenance. Although current methods permit accurate comparisons of the effects of molecular manipulations and drug applications on cell motility, effects of alterations in subcellular activities on motility cannot be fully elucidated from those methods. Here, we develop a strategy termed cell-nuclear (CN) correlation to parameterize represented dynamic subcellular activities and to quantify their contributions in mesenchymal-like migration. Based on the biophysical meaning of the CN correlation, we propose a cell migration potential index (CMPI) to measure cell motility. When the effectiveness of CMPI was evaluated with respect to one of the most popular cell migration analysis methods, Persistent Random Walk, we found that the cell motility estimates among six cell lines used in this study were highly consistent between these two approaches. Further evaluations indicated that CMPI can be determined using a shorter time period and smaller cell sample size, and it possesses excellent reliability and applicability, even in the presence of a wide range of noise, as might be generated from individual imaging acquisition systems. The novel approach outlined here introduces a robust strategy through an analysis of subcellular locomotion activities for single cell migration assessment.

  12. Dephosphorylation of Major Sperm Protein (MSP) Fiber Protein 3 by Protein Phosphatase 2A during Cell Body Retraction in the MSP-based Amoeboid Motility of Ascaris Sperm

    PubMed Central

    Yi, Kexi; Wang, Xu; Emmett, Mark R.; Marshall, Alan G.; Stewart, Murray

    2009-01-01

    The crawling movement of nematode sperm requires coordination of leading edge protrusion with cell body retraction, both of which are powered by modulation of a cytoskeleton based on major sperm protein (MSP) filaments. We used a cell-free in vitro motility system in which both protrusion and retraction can be reconstituted, to identify two proteins involved in cell body retraction. Pharmacological and depletion-add back assays showed that retraction was triggered by a putative protein phosphatase 2A (PP2A, a Ser/Thr phosphatase activated by tyrosine dephosphorylation). Immunofluorescence showed that PP2A was present in the cell body and was concentrated at the base of the lamellipod where the force for retraction is generated. PP2A targeted MSP fiber protein 3 (MFP3), a protein unique to nematode sperm that binds to the MSP filaments in the motility apparatus. Dephosphorylation of MFP3 caused its release from the cytoskeleton and generated filament disassembly. Our results suggest that interaction between PP2A and MFP3 leads to local disassembly of the MSP cytoskeleton at the base of the lamellipod in sperm that in turn pulls the trailing cell body forward. PMID:19458186

  13. Nonrigid Registration of 2-D and 3-D Dynamic Cell Nuclei Images for Improved Classification of Subcellular Particle Motion

    PubMed Central

    Kim, Il-Han; Chen, Yi-Chun M.; Spector, David L.; Eils, Roland; Rohr, Karl

    2012-01-01

    The observed motion of subcellular particles in fluorescence microscopy image sequences of live cells is generally a superposition of the motion and deformation of the cell and the motion of the particles. Decoupling the two types of movements to enable accurate classification of the particle motion requires the application of registration algorithms. We have developed an intensity-based approach for nonrigid registration of multi-channel microscopy image sequences of cell nuclei. First, based on 3-D synthetic images we demonstrate that cell nucleus deformations change the observed motion types of particles and that our approach allows to recover the original motion. Second, we have successfully applied our approach to register 2-D and 3-D real microscopy image sequences. A quantitative experimental comparison with previous approaches for nonrigid registration of cell microscopy has also been performed. PMID:20840894

  14. Regulation of calnexin sub-cellular localization modulates endoplasmic reticulum stress-induced apoptosis in MCF-7 cells.

    PubMed

    Delom, Frédéric; Fessart, Delphine; Chevet, Eric

    2007-02-01

    The endoplasmic reticulum (ER) is the cellular compartment where proteins enter the secretory pathway, undergo post-translational modifications and acquire a correct conformation. If these functions are chronically altered, specific ER stress signals are triggered to promote cell death through the intrinsic apoptotic pathway. Here, we show that tunicamycin causes significant alteration of calnexin sub-cellular distribution in MCF-7 cells. Interestingly, this correlates with the absence of both tunicamycin-induced calnexin phosphorylation as well as tunicamycin-induced cell death. Under these conditions, calnexin-associated Bap31, an ER integral membrane protein, is subjected to a caspase-8 cleavage pattern within a specific sub-compartment of the ER. These results suggest that MCF-7 resistance to ER stress-induced apoptosis is partially mediated by the expression level of calnexin which in turn controls its sub-cellular localization, and its association with Bap31. These data may delineate a resistance mechanism to the ER stress-induced intrinsic apoptotic pathway.

  15. The Effects of Cell Phone Waves (900 MHz-GSM Band) on Sperm Parameters and Total Antioxidant Capacity in Rats

    PubMed Central

    Ghanbari, Masoud; Mortazavi, Seyed Bagher; Khavanin, Ali; Khazaei, Mozafar

    2013-01-01

    Background: There is tremendous concern regarding the possible adverse effects of cell phone microwaves. Contradictory results, however, have been reported for the effects of these waves on the body. In the present study, the effect of cell phone microwaves on sperm parameters and total antioxidant capacity was investigated with regard to the duration of exposure and the frequency of these waves. Materials and Methods: This experimental study was performed on 28 adult male Wistar rats (200-250 g). The animals were randomly assigned to four groups (n=7): i. control; ii. two-week exposure to cell phone-simulated waves; iii. three-week exposure to cell phonesimulated waves; and iv. two-week exposure to cell phone antenna waves. In all groups, sperm analysis was performed based on standard methods and we determined the mean sperm total antioxidant capacity according to the ferric reducing ability of plasma (FRAP) method. Data were analyzed by one-way ANOVA followed by Tukey’s test using SPSS version 16 software. Results: The results indicated that sperm viability, motility, and total antioxidant capacity in all exposure groups decreased significantly compared to the control group (p<0.05). Increasing the duration of exposure from 2 to 3 weeks caused a statistically significant decrease in sperm viability and motility (p<0.05). Conclusion: Exposure to cell phone waves can decrease sperm viability and motility in rats. These waves can also decrease sperm total antioxidant capacity in rats and result in oxidative stress. PMID:24520459

  16. Sex determination. foxl3 is a germ cell-intrinsic factor involved in sperm-egg fate decision in medaka.

    PubMed

    Nishimura, Toshiya; Sato, Tetsuya; Yamamoto, Yasuhiro; Watakabe, Ikuko; Ohkawa, Yasuyuki; Suyama, Mikita; Kobayashi, Satoru; Tanaka, Minoru

    2015-07-17

    Sex determination is an essential step in the commitment of a germ cell to a sperm or egg. However, the intrinsic factors that determine the sexual fate of vertebrate germ cells are unknown. Here, we show that foxl3, which is expressed in germ cells but not somatic cells in the gonad, is involved in sperm-egg fate decision in medaka fish. Adult XX medaka with disrupted foxl3 developed functional sperm in the expanded germinal epithelium of a histologically functional ovary. In chimeric medaka, mutant germ cells initiated spermatogenesis in female wild-type gonad. These results indicate that a germ cell-intrinsic cue for the sperm-egg fate decision is present in medaka and that spermatogenesis can proceed in a female gonadal environment.

  17. Geometry-Specific Heterogeneity of the Apparent Diffusion Rate of Materials Inside Sperm Cells

    PubMed Central

    Takao, Daisuke; Kamimura, Shinji

    2010-01-01

    Abstract In sea urchin spermatozoa, the energy source powering flagellar motion is provided as ATP produced by mitochondria located at the proximal ends of flagella. However, the bottleneck structure between the sperm head and the flagellar tail seems to restrict the free entry of ATP from mitochondria into the tail region. To test this possibility, we investigated the diffusion properties in sperm cells using fluorescence recovery after photobleaching. We found that the rate of fluorescence recovery in the head region was ∼10% of that observed in the flagellar tail regions. We also found that, even within the tail region, rates varied depending on location, i.e., rates were slower at the more distal regions. Using computational analysis, the rate heterogeneity was shown to be caused mainly by the geometry of the sperm structure, even if little or no difference in diffusion rates through the neck region was assumed. Therefore, we concluded that materials such as ATP would generally diffuse freely between the heads and the flagella of sperm cells. We believe these findings regarding the diffusion properties inside spermatozoa provide further insights into material transportation and chemical signaling inside eukaryotic cilia and flagella. PMID:20409478

  18. Geometry-specific heterogeneity of the apparent diffusion rate of materials inside sperm cells.

    PubMed

    Takao, Daisuke; Kamimura, Shinji

    2010-04-21

    In sea urchin spermatozoa, the energy source powering flagellar motion is provided as ATP produced by mitochondria located at the proximal ends of flagella. However, the bottleneck structure between the sperm head and the flagellar tail seems to restrict the free entry of ATP from mitochondria into the tail region. To test this possibility, we investigated the diffusion properties in sperm cells using fluorescence recovery after photobleaching. We found that the rate of fluorescence recovery in the head region was approximately 10% of that observed in the flagellar tail regions. We also found that, even within the tail region, rates varied depending on location, i.e., rates were slower at the more distal regions. Using computational analysis, the rate heterogeneity was shown to be caused mainly by the geometry of the sperm structure, even if little or no difference in diffusion rates through the neck region was assumed. Therefore, we concluded that materials such as ATP would generally diffuse freely between the heads and the flagella of sperm cells. We believe these findings regarding the diffusion properties inside spermatozoa provide further insights into material transportation and chemical signaling inside eukaryotic cilia and flagella.

  19. Label-free biochemical characterization of bovine sperm cells using Raman microscopy

    NASA Astrophysics Data System (ADS)

    De Luca, A. C.; Manago, S.; Ferrara, M. A.; Sirleto, L.; Puglisi, R.; Balduzzi, D.; Galli, A.; Rendina, I.; Ferraro, P.; Coppola, G.

    2013-04-01

    Raman spectroscopy is a label-free and non-invasive method that measures the inelastic scattered light from a sample giving insight into the vibration eigenmodes of the excited molecules. For these reasons, Raman spectroscopy has been used as a powerful tool to investigate different biological tissues and living cells. In this paper, we present a Raman spectroscopy-based method for sensitive biochemical characterization of bovine sperm cells. Importantly, by analysing separate Raman spectra from the nucleus, acrosomale vesicle and tail of single sperm cells, we are able to identify characteristic Raman features associated with DNA, protein and lipid molecular vibrations for discriminating among different locations inside the cell with sub-micrometric resolution (˜0.3 μm). We demonstrate that our Raman spectroscopy facilitates spectral assignment and increases detection sensitivity, opening the way for novel bio-imaging platforms.

  20. Differentiation of zebrafish spermatogonial stem cells to functional sperm in culture.

    PubMed

    Kawasaki, Toshihiro; Siegfried, Kellee R; Sakai, Noriyoshi

    2016-02-15

    Molecular dissection and chemical screening on a complex process such as spermatogenesis could be facilitated by cell culture approaches that allow easy access for experimental manipulation and live imaging of specific molecules; however, technical limitations have thus far prevented the complete reconstruction of spermatogenic events in cell culture. Here, we describe the production of functional sperm from self-renewing spermatogonial stem cells (SSCs) in cell culture conditions, using zebrafish testicular hyperplasia cells that accumulate early stage spermatogonia. By serially transplanting hyperplasias into immunodeficient rag1 mutant zebrafish, we succeeded in long-term maintenance and efficient production of starting material for SSC culture. Through improvements of culture conditions, we achieved efficient propagation of SSCs derived from the hyperplasia. When SSCs that underwent the SSC-propagating step for 1 month were transferred onto Sertoli feeder cells, they differentiated into functional sperm that gave rise to offspring. Oxygen at the concentration of air proved to be detrimental for sperm differentiation from SSCs, but not for propagation of SSCs. These results indicate that the whole spermatogenic process can be represented in cell culture in zebrafish, facilitating analyses of the molecular mechanisms of spermatogenesis in vertebrates.

  1. Generation of rats from vitrified oocytes with surrounding cumulus cells via in vitro fertilization with cryopreserved sperm.

    PubMed

    Fujiwara, Katsuyoshi; Kamoshita, Maki; Kato, Tsubasa; Ito, Junya; Kashiwazaki, Naomi

    2017-01-01

    The objective of this study was to evaluate fertility and full-term development of rat vitrified oocytes after in vitro fertilization (IVF) with cryopreserved sperm. Oocytes with or without surrounding cumulus cells were vitrified with 30% ethylene glycol + 0.5 mol/L sucrose + 20% fetal calf serum by using the Cryotop method. The warmed oocytes were co-cultured with sperm. Although the denuded/vitrified oocytes were not fertilized, some of the oocytes vitrified with cumulus cells were fertilized (32.7%) after IVF with fresh sperm. When IVF was performed with cryopreserved sperm, vitrified or fresh oocytes with cumulus cells were fertilized (62.9% or 41.1%, respectively). In addition, to confirm the full-term development of the vitrified oocytes with surrounding cumulus cells after IVF with cryopreserved sperm, 108 vitrified oocytes with two pronuclei (2PN) were transferred into eight pseudopregnant females, and eight pups were obtained from three recipients. The present work demonstrates that vitrified rat oocytes surrounded by cumulus cells can be fertilized in vitro with cryopreserved sperm, and that 2PN embryos derived from cryopreserved gametes can develop to term. To our knowledge, this is the first report of successful generation of rat offspring derived from vitrified oocytes that were fertilized in vitro with cryopreserved sperm.

  2. Receptor tyrosine kinase EphA7 is required for interneuron connectivity at specific subcellular compartments of granule cells

    PubMed Central

    Beuter, Simone; Ardi, Ziv; Horovitz, Omer; Wuchter, Jennifer; Keller, Stefanie; Saha, Rinki; Tripathi, Kuldeep; Anunu, Rachel; Kehat, Orli; Kriebel, Martin; Richter-Levin, Gal; Volkmer, Hansjürgen

    2016-01-01

    Neuronal transmission is regulated by the local circuitry which is composed of principal neurons targeted at different subcellular compartments by a variety of interneurons. However, mechanisms that contribute to the subcellular localisation and maintenance of GABAergic interneuron terminals are poorly understood. Stabilization of GABAergic synapses depends on clustering of the postsynaptic scaffolding protein gephyrin and its interaction with the guanine nucleotide exchange factor collybistin. Lentiviral knockdown experiments in adult rats indicated that the receptor tyrosine kinase EphA7 is required for the stabilisation of basket cell terminals on proximal dendritic and somatic compartments of granular cells of the dentate gyrus. EphA7 deficiency and concomitant destabilisation of GABAergic synapses correlated with impaired long-term potentiation and reduced hippocampal learning. Reduced GABAergic innervation may be explained by an impact of EphA7 on gephyrin clustering. Overexpression or ephrin stimulation of EphA7 induced gephyrin clustering dependent on the mechanistic target of rapamycin (mTOR) which is an interaction partner of gephyrin. Gephyrin interactions with mTOR become released after mTOR activation while enhanced interaction with the guanine nucleotide exchange factor collybistin was observed in parallel. In conclusion, EphA7 regulates gephyrin clustering and the maintenance of inhibitory synaptic connectivity via mTOR signalling. PMID:27405707

  3. Antibodies to spermatozoa. III. Responses in rabbits and guinea-pigs to immunization with guinea-pig sperm cells

    PubMed Central

    Hekman, Annemarie; Shulman, S.

    1971-01-01

    The antigens of guinea-pig sperm cells, of both the epididymal and ejaculated (or seminal) types, have been studied, using rabbit and guinea-pig antisera. Several antigens could be revealed by gel diffusion studies, using well-washed but non-ruptured sperm cells, indicating that intentional cell breaking is not essential for demonstrating the antigens. This release of soluble antigen was followed as a function of time and temperature, both as total protein in supernatants and in increasing strength of precipitation. With rabbit antiserum, epididymal sperm showed two antigens, that were also demonstrated in epididymal and testicular extract and in seminal sperm. These other materials revealed additional antigens with these antisera. Immunofluorescent staining was limited to the acrosomes. With guinea-pig antibodies, no precipitating antigen that was characteristic of sperm could be seen. These antisera showed immunofluorescent staining of the acrosomes. The staining could be distinguished, in terms of thermostability, from the staining produced by normal serum. No evidence was found for the occurrence of any sperm-coating antigens in the guinea-pig, especially since both antiseminal plasma and antivesicular fluid antisera failed to give immunofluorescent staining of the sperm cells. ImagesFig. 1Fig. 3Fig. 4Fig. 5Fig. 6 PMID:4998924

  4. Involvement of redox- and phosphorylation-dependent pathways in osmotic adaptation in sperm cells of euryhaline tilapia.

    PubMed

    Morita, Masaya; Nakajima, Ayako; Takemura, Akihiro; Okuno, Makoto

    2011-06-15

    Sperm cells involved in fertilisation must tolerate hypo-osmotic and hyper-osmotic environments. Euryhaline tilapia (Oreochromis mossambicus) can acclimatise to and reproduce in freshwater and seawater because its sperm are able to adapt to these differing osmotic environments. In this study, we found that the dephosphorylation of sperm proteins in O. mossambicus correlated with the activation of flagellar motility when sperm were exposed to hypotonic or hypertonic conditions, and that differences in phosphorylation may reflect adaptations to a given osmotic environment. Of the sperm proteins that were dephosphorylated, the phosphorylation pattern of an 18 kDa protein, identified as the superoxide anion scavenger Cu/Zn superoxide dismutase (Cu/Zn SOD), was different in freshwater- and seawater-acclimatised tilapia sperm. Cu/Zn SOD was distributed from the sperm head to the flagellum. Additionally, differences were observed between freshwater and seawater tilapia in the nitration of tyrosine residues (which might be mediated by SOD) in sperm flagellar proteins in response to osmotic shock. These results demonstrate that reactive-oxygen-species-dependent mechanisms contribute to both osmotic tolerance and the activation of flagellar motility.

  5. Cellular Distribution and Subcellular Localization of Molecular Components of Vesicular Transmitter Release in Horizontal Cells of Rabbit Retina

    PubMed Central

    HIRANO, ARLENE A.; BRANDSTÄTTER, JOHANN H.; BRECHA, NICHOLAS C.

    2010-01-01

    The mechanism underlying transmitter release from retinal horizontal cells is poorly understood. We investigated the possibility of vesicular transmitter release from mammalian horizontal cells by examining the expression of synaptic proteins that participate in vesicular transmitter release at chemical synapses. Using immunocytochemistry, we evaluated the cellular and subcellular distribution of complexin I/II, syntaxin-1, and synapsin I in rabbit retina. Strong labeling for complexin I/II, proteins that regulate a late step in vesicular transmitter release, was found in both synaptic layers of the retina, and in somata of A- and B-type horizontal cells, of γ-aminobutyric acid (GABA)- and glycinergic amacrine cells, and of ganglion cells. Immunoelectron microscopy demonstrated the presence of complexin I/II in horizontal cell processes postsynaptic to rod and cone ribbon synapses. Syntaxin-1, a core protein of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex known to bind to complexin, and synapsin I, a synaptic vesicle-associated protein involved in the Ca2+-dependent recruitment of synaptic vesicles for transmitter release, were also present in the horizontal cells and their processes at photoreceptor synapses. Photoreceptors and bipolar cells did not express any of these proteins at their axon terminals. The presence of complexin I/II, syntaxin-1, and synapsin I in rabbit horizontal cell processes and tips suggests that a vesicular mechanism may underlie transmitter release from mammalian horizontal cells. PMID:15912504

  6. New application of a subcellular fractionation method to kidney and testis for the determination of conjugated linoleic acid in selected cell organelles of healthy and cancerous human tissues.

    PubMed

    Hoffmann, Kristina; Blaudszun, Jörg; Brunken, Claus; Höpker, Wilhelm-Wolfgang; Tauber, Roland; Steinhart, Hans

    2005-03-01

    To clarify the mechanism of the anticarcinogenic effect of conjugated linoleic acid (CLA), its intracellular distribution needs to be determined. Subcellular fractionation using centrifugation techniques is a method that is frequently used for isolation of cell organelles from different tissues. But as the size and density of the organelles differ, the method needs to be optimised for every type of tissue. The novelty of this study is the application of a subcellular fractionation method to human healthy and cancerous renal and testicular tissue. Separation of total tissue homogenate into nuclei, cytosol, and a mixture of mitochondria and plasma membranes was achieved by differential centrifugation. As mitochondria and plasma membranes seemed to be too similar in size and weight to be separated by differential centrifugation, discontinuous density-gradient centrifugation was carried out successfully. The purity of the subcellular fractions was checked by measuring the activity of marker enzymes. All fractions were highly enriched in their corresponding marker enzyme. However, the nuclear fractions of kidney and renal cell carcinoma were slightly contaminated with mitochondria and plasma membrane fractions of all tissues with lysosomes. The fraction designated the cytosolic fraction contained not only cytosol, but also microsomes and lysosomes. The CLA contents of the subcellular fractions were in the range 0.13-0.37% of total fatty acids and were lowest in the plasma membrane fractions of all types of tissue studied. C16:0, C18:0, C18:1 c9, C18:2 n-6, and C20:4 n-6 were found to be the major fatty acids in all the subcellular fractions studied. However, marked variations in fatty acid content between subcellular fractions and between types of tissue were detectable. Because of these differences between tissues, no general statement on characteristic fatty acid profiles of single subcellular fractions is possible.

  7. Study of acetowhitening mechanisms in live mammalian cells with label-free subcellular-level multimodal nonlinear optical microscopy

    NASA Astrophysics Data System (ADS)

    Lin, Jian; Teh, Sengkhoon; Zheng, Wei; Wang, Zi; Huang, Zhiwei

    2015-03-01

    The tissue acetowhitening effect in acetic acid instillation procedure is a simple and economic method for neoplasia detection and has been clinically utilized since 1925. It is suspected that the optical property (e.g. scattering) change in acetowhitening is due to coagulation of intracellular proteins, but no experimental proof has been reported yet. In this work, we use third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) to investigate the acetowhitening phenomenon induced by acidic acid in live mammalian cells without labeling. We studied the acetowhitening effect with different acetic acid concentrations and the co-localized TPEF and THG imaging on tryptophan and NADH at subcellular-level reveals that the acetowhitening phenomenon is highly related with proteins involved in metabolic pathways in the nucleus and cytoplasm in live cells.

  8. Habits of cell phone usage and sperm quality - does it warrant attention?

    PubMed

    Zilberlicht, Ariel; Wiener-Megnazi, Zofnat; Sheinfeld, Yulia; Grach, Bronislava; Lahav-Baratz, Shirly; Dirnfeld, Martha

    2015-09-01

    Male infertility constitutes 30-40% of all infertility cases. Some studies have shown a continuous decline in semen quality since the beginning of the 20th century. One postulated contributing factor is radio frequency electromagnetic radiation emitted from cell phones. This study investigates an association between characteristics of cell phone usage and semen quality. Questionnaires accessing demographic data and characteristics of cell phone usage were completed by 106 men referred for semen analysis. Results were analysed according to WHO 2010 criteria. Talking for ≥1 h/day and during device charging were associated with higher rates of abnormal semen concentration (60.9% versus 35.7%, P < 0.04 and 66.7% versus 35.6%, P < 0.02, respectively). Among men who reported holding their phones ≤50 cm from the groin, a non-significantly higher rate of abnormal sperm concentration was found (47.1% versus 11.1%). Multivariate analysis revealed that talking while charging the device and smoking were risk factors for abnormal sperm concentration (OR = 4.13 [95% CI 1.28-13.3], P < 0.018 and OR = 3.04 [95% CI 1.14-8.13], P < 0.027, respectively). Our findings suggest that certain aspects of cell phone usage may bear adverse effects on sperm concentration. Investigation using large-scale studies is thus needed.

  9. Microfluidics and numerical simulation as methods for standardization of zebrafish sperm cell activation.

    PubMed

    Scherr, Thomas; Knapp, Gerald L; Guitreau, Amy; Park, Daniel Sang-Won; Tiersch, Terrence; Nandakumar, Krishnaswamy; Monroe, W Todd

    2015-01-01

    Sperm cell activation plays a critical role in a range of biological and engineering processes, from fertilization to cryopreservation protocol evaluation. Across a range of species, ionic and osmotic effects have been discovered that lead to activation. Sperm cells of zebrafish (Danio rerio) initiate motility in a hypoosmotic environment. In this study, we employ a microfluidic mixer for the purpose of rapidly diluting the extracellular medium to initiate the onset of cell motility. The use of a microchannel offers a rapid and reproducible mixing profile throughout the device. This greatly reduces variability from trial to trial relative to the current methods of analysis. Coupling these experiments with numerical simulations, we were able to investigate the dynamics of intracellular osmolality as each cell moves along its path through the micromixer. Our results suggest that intracellular osmolality, and hence intracellular ion concentration, only slightly decreases, contrary to the common thought that larger changes in these parameters are required for activation. Utilizing this framework, microfluidics for controlled extracellular environments and associated numerical modeling, has practical applicability in standardizing high-throughput aquatic sperm activation, and more fundamentally, investigations of the intracellular environment leading to motility.

  10. Subcellular distribution of the 18kDa translocator protein and transcript variant PBR-S in human cells.

    PubMed

    Liu, Guo-Jun; Middleton, Ryan J; Banati, Richard B

    2017-05-20

    Despite continued interest in the 18kDa translocator protein (PBR/TSPO) as a biomarker and a therapeutic target for a range of diseases, its functional role, such as in the steroid synthesis pathway and energy metabolism has either become contentious or remains to be described more precisely. The PBR/TSPO gene consists of four exons, while a shorter isoform termed PBR-S lacks exon 2. The PBR-S 102-codon open reading frame differs to that of PBR/TSPO, resulting in a protein that is completely unrelated to PBR/TSPO. To our knowledge, PBR-S protein has never been described and has no known or proposed function. To obtain possible clues on the role of this uncharacterised protein, we compared the subcellular distribution of PBR-S to that of PBR/TSPO. By expressing fluorescently tagged PBR/TSPO, we confirmed that full-length PBR/TSPO co-localises with mitochondria in HeLa, HEK-293, MDA-MB-231, BJ and U87-MG human cell lines. Unlike the strictly mitochondrial localisation of PBR/TSPO, PBR-S has a punctate distribution throughout the cytosol that co-localises with lysosomes in HeLa, HEK-293, MDA-MB-231, BJ and U87-MG cells. In summary, within the cell lines examined we confirm mitochondria rather than occasionally reported other localisations, such as the cell nucleus, to be the only site where PBR/TSPO resides. Due to the lack of any shared protein sequences and the different subcellular locations, we suggest that the previously uncharacterised PBR-S protein variant of the PBR/TSPO gene is likely to serve a different yet to be discovered function compared to PBR/TSPO.

  11. Biochemical and topological analysis of bovine sperm cells induced by low power laser irradiation

    NASA Astrophysics Data System (ADS)

    Dreyer, T. R.; Siqueira, A. F. P.; Magrini, T. D.; Fiorito, P. A.; Assumpção, M. E. O. A.; Nichi, M.; Martinho, H. S.; Milazzotto, M. P.

    2011-07-01

    Low-level laser irradiation (LLLI) increases ATP production and energy supply to the cell which could increase sperm motility, acrossomal reaction and consequently the fertilizing potential. The aim of this study was to characterize the biochemical and topological changes induced by low power laser irradiation on bull sperm cells. Post-thawing sperm were irradiated with a 633nm laser with fluence rates of 30, 150 and 300mJ.cm-2 (power of 5mW for 1, 5 and 10minutes, respectively); 45, 230, and 450mJ.cm-2 (7.5mW for 1, 5 and 10 minutes); and 60, 300 and 600mJ.cm-2 (10mW for 1, 5 and 10 minutes). Biochemical and metabolical changes were analyzed by FTIR and flow cytometry; oxygen reactive species production was assessed by TBARS and the morphological changes were evaluated by AFM. Motility had no difference among times or powers of irradiation. Increasing in ROS generation was observed with power of 5mW compared to 7.5 and 10mW, and with 10min of irradiation in comparison with 5 and 1min of irradiation. This higher ROS generation was related to an increase in acrossomal and plasma membrane damage. FTIR results showed that the amount of lipids was inversely proportional to the quantity of ROS generated. AFM images showed morphological differences in plasma/acrossomal membrane, mainly on the equatorial region. We conclude that LLLI is an effective method to induce changes on sperm cell metabolism but more studies are necessary to establish an optimal dose to increase the fertility potential of these cells.

  12. Long-term effects of triethylenemelamine exposure on mouse testis cells and sperm chromatin structure assayed by flow cytometry

    SciTech Connect

    Evenson, D.P.; Baer, R.K.; Jost, L.K. )

    1989-01-01

    The toxic and potentially mutagenic actions of triethylenemelamine (TEM) on mouse body and testis weights, testicular cell kinetics, sperm production, sperm head morphology, and sperm chromatin structure were assessed in two experiments. The first experiment examined effects of four dose levels of TEM, assayed 1, 4, or 10 wk after toxic exposure. In the second study, effects from five dosage levels were measured at 1, 4, and 10 wk, and the highest dosage level was evaluated over 44 wk. TEM produced an expected dose related loss of spermatogenic activity and subsequent recovery as determined by dual-parameter (DNA, RNA) flow cytometry (FCM) measurements of testicular cells. Both testicular weights and caudal sperm reserves remained generally below controls after 44 wk recovery following exposure to the highest dosage. Chromatin structure alterations, defined as increased susceptibility to DNA denaturation in situ, and sperm head morphology were highly correlated with dose and with each other. Sperm head morphology and sperm chromatic structure remained abnormal at 44 wk for the 1.0 mg/kg TEM dosage, suggesting that the abnormalities, present long after the initial toxic response, may be a result of mutation. This study demonstrates that flow cytometry provides a unique, rapid, and efficient means to measure effects of reproductive toxins and potential mutagens.

  13. Spectrin βV adaptive mutations and changes in subcellular location correlate with emergence of hair cell electromotility in mammalians.

    PubMed

    Cortese, Matteo; Papal, Samantha; Pisciottano, Francisco; Elgoyhen, Ana Belén; Hardelin, Jean-Pierre; Petit, Christine; Franchini, Lucia Florencia; El-Amraoui, Aziz

    2017-02-21

    The remarkable hearing capacities of mammals arise from various evolutionary innovations. These include the cochlear outer hair cells and their singular feature, somatic electromotility, i.e., the ability of their cylindrical cell body to shorten and elongate upon cell depolarization and hyperpolarization, respectively. To shed light on the processes underlying the emergence of electromotility, we focused on the βV giant spectrin, a major component of the outer hair cells' cortical cytoskeleton. We identified strong signatures of adaptive evolution at multiple sites along the spectrin-βV amino acid sequence in the lineage leading to mammals, together with substantial differences in the subcellular location of this protein between the frog and the mouse inner ear hair cells. In frog hair cells, spectrin βV was invariably detected near the apical junctional complex and above the cuticular plate, a dense F-actin meshwork located underneath the apical plasma membrane. In the mouse, the protein had a broad punctate cytoplasmic distribution in the vestibular hair cells, whereas it was detected in the entire lateral wall of cochlear outer hair cells and had an intermediary distribution (both cytoplasmic and cortical, but restricted to the cell apical region) in cochlear inner hair cells. Our results support a scenario where the singular organization of the outer hair cells' cortical cytoskeleton may have emerged from molecular networks initially involved in membrane trafficking, which were present near the apical junctional complex in the hair cells of mammalian ancestors and would have subsequently expanded to the entire lateral wall in outer hair cells.

  14. Transmembrane topology, subcellular distribution and turnover of the gamma-aminobutyric acid/benzodizaepine receptor in chick brain cell cultures

    SciTech Connect

    Czajkowski, C.M.

    1987-01-01

    Experiments were performed utilizing trypsinization of the GABA/BZD-R in intact cells to determine (1) the subcellular distribution of membrane-associated GABA/BZD-Rs and (2) aspects of the transmembrane topology of the BZD-R. Additionally, R07-0213, a positively charged benzodiazepine, was used to distinguish between cell surface and intracellular BZD-Rs. Following trypsin treatment of intact cells a cleaved receptor fragment of M{sub r} = 24,000 (xRF24) is generated. It remains anchored in the plasma membrane and not only retains the ability to bind ({sup 3}H)flunitrazepan reversibly and irreversibly but also retains the ability to be modulated by GABA. xRF24 is not observed following trypsinization of saponin-treated cells or cell homogenates, indicating that it has a cytoplasmic domain as well as a cell surface domain, as expected for a transmembrane fragment of the BZD-R. By utilizing ({sup 3}H)flunitrazepam as an irreversible photoaffinity label, BZD-R turnover was also investigated.

  15. Cytochrome c upregulation during capacitation and spontaneous acrosome reaction determines the fate of pig sperm cells: linking proteome analysis.

    PubMed

    Choi, Yun-Jung; Uhm, Sang-Jun; Song, Sang-Jin; Song, Hyuk; Park, Jin-Ki; Kim, Teoan; Park, Chankyu; Kim, Jin-Hoi

    2008-02-01

    To identify the mechanisms underlying capacitation, we undertook a high-resolution differential proteomic analysis of pig sperm cells. Two-dimensional gel electrophoresis and subsequent MALDI-TOF mass spectrometry analyses led to identification of 56 differentially expressed proteins. After induction of capacitation in vitro, the well-established markers of the capacitation (lactadherin P47, acrosomal protein SP-10 precursor, prohibitin, proteasomes, DJ-1 protein and arylsulfatase-A) and TCA cycle proteins (isocitrate dehydrogenase, malate dehydrogenase and pyruvate dehydrogenase) were identified. During induction, cytochrome c expression via the p53 pathway increased, however apoptotic executors, such as caspase-3, decreased significantly. Therefore, we tested the hypothesis that cytochrome c upregulation in spermatozoa is capable of activating tyrosine phosphorylation for capacitation, rather than apoptosis. Exposure of sperm cells to soluble Na2CrO4 [Cr (VI)], which induces cytochrome c upregulation, caused a dose- and time-dependent increase in tyrosine phosphorylation of sperm proteins in non-capacitating medium. In contrast, supplementation of cyclosporin A, which blocks cytochrome c upregulation, inhibited tyrosine phosphorylation of sperm proteins. Furthermore, spermatozoa in capacitation medium or non-capacitation media supplemented with soluble Cr (VI) showed similar levels of capacitation. These findings indicate that differential expression of many of these proteins has previously been unrecognized in sperm cells incubated in capacitation medium also suggest that a gradual increase of cytochrome c during incubation to induce capacitation determines sperm cell fate, i.e., apoptosis or further development for fertilization.

  16. Spectrin βV adaptive mutations and changes in subcellular location correlate with emergence of hair cell electromotility in mammalians

    PubMed Central

    Cortese, Matteo; Papal, Samantha; Pisciottano, Francisco; Elgoyhen, Ana Belén; Hardelin, Jean-Pierre; Petit, Christine; Franchini, Lucia Florencia; El-Amraoui, Aziz

    2017-01-01

    The remarkable hearing capacities of mammals arise from various evolutionary innovations. These include the cochlear outer hair cells and their singular feature, somatic electromotility, i.e., the ability of their cylindrical cell body to shorten and elongate upon cell depolarization and hyperpolarization, respectively. To shed light on the processes underlying the emergence of electromotility, we focused on the βV giant spectrin, a major component of the outer hair cells' cortical cytoskeleton. We identified strong signatures of adaptive evolution at multiple sites along the spectrin-βV amino acid sequence in the lineage leading to mammals, together with substantial differences in the subcellular location of this protein between the frog and the mouse inner ear hair cells. In frog hair cells, spectrin βV was invariably detected near the apical junctional complex and above the cuticular plate, a dense F-actin meshwork located underneath the apical plasma membrane. In the mouse, the protein had a broad punctate cytoplasmic distribution in the vestibular hair cells, whereas it was detected in the entire lateral wall of cochlear outer hair cells and had an intermediary distribution (both cytoplasmic and cortical, but restricted to the cell apical region) in cochlear inner hair cells. Our results support a scenario where the singular organization of the outer hair cells’ cortical cytoskeleton may have emerged from molecular networks initially involved in membrane trafficking, which were present near the apical junctional complex in the hair cells of mammalian ancestors and would have subsequently expanded to the entire lateral wall in outer hair cells. PMID:28179572

  17. Expression of temperature-sensitive ion channel TRPM8 in sperm cells correlates with vertebrate evolution

    PubMed Central

    Majhi, Rakesh Kumar; Saha, Somdatta; Kumar, Ashutosh; Ghosh, Arijit; Swain, Nirlipta; Goswami, Luna; Mohapatra, Pratyush; Maity, Apratim; Kumar Sahoo, Vivek

    2015-01-01

    Transient Receptor Potential cation channel, subfamily Melastatin, member 8 (TRPM8) is involved in detection of cold temperature, different noxious compounds and in execution of thermo- as well as chemo-sensitive responses at cellular levels. Here we explored the molecular evolution of TRPM8 by analyzing sequences from various species. We elucidate that several regions of TRPM8 had different levels of selection pressure but the 4th–5th transmembrane regions remain highly conserved. Analysis of synteny suggests that since vertebrate origin, TRPM8 gene is linked with SPP2, a bone morphogen. TRPM8, especially the N-terminal region of it, seems to be highly variable in human population. We found 16,656 TRPM8 variants in 1092 human genomes with top variations being SNPs, insertions and deletions. A total of 692 missense mutations are also mapped to human TRPM8 protein of which 509 seem to be delateroiours in nature as supported by Polyphen V2, SIFT and Grantham deviation score. Using a highly specific antibody, we demonstrate that TRPM8 is expressed endogenously in the testis of rat and sperm cells of different vertebrates ranging from fish to higher mammals. We hypothesize that TRPM8 had emerged during vertebrate evolution (ca 450 MYA). We propose that expression of TRPM8 in sperm cell and its role in regulating sperm function are important factors that have guided its molecular evolution, and that these understandings may have medical importance. PMID:26500819

  18. An EAR-Dependent Regulatory Module Promotes Male Germ Cell Division and Sperm Fertility in Arabidopsis.

    PubMed

    Borg, Michael; Rutley, Nicholas; Kagale, Sateesh; Hamamura, Yuki; Gherghinoiu, Mihai; Kumar, Sanjeev; Sari, Ugur; Esparza-Franco, Manuel A; Sakamoto, Wataru; Rozwadowski, Kevin; Higashiyama, Tetsuya; Twell, David

    2014-05-01

    The production of the sperm cells in angiosperms requires coordination of cell division and cell differentiation. In Arabidopsis thaliana, the germline-specific MYB protein DUO1 integrates these processes, but the regulatory hierarchy in which DUO1 functions is unknown. Here, we identify an essential role for two germline-specific DUO1 target genes, DAZ1 and DAZ2, which encode EAR motif-containing C2H2-type zinc finger proteins. We show that DAZ1/DAZ2 are required for germ cell division and for the proper accumulation of mitotic cyclins. Importantly, DAZ1/DAZ2 are sufficient to promote G2- to M-phase transition and germ cell division in the absence of DUO1. DAZ1/DAZ2 are also required for DUO1-dependent cell differentiation and are essential for gamete fusion at fertilization. We demonstrate that the two EAR motifs in DAZ1/DAZ2 mediate their function in the male germline and are required for transcriptional repression and for physical interaction with the corepressor TOPLESS. Our findings uncover an essential module in a regulatory hierarchy that drives mitotic transition in male germ cells and implicates gene repression pathways in sperm cell formation and fertility.

  19. An ARID domain-containing protein within nuclear bodies is required for sperm cell formation in Arabidopsis thaliana

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In plants, each male meiotic product undergoes mitosis, and then one of the resulting cells divides again, yielding a three-celled pollen grain comprised of a vegetative cell and two sperm cells. Several genes have been found to act in this process, and DUO1 (DUO POLLEN 1), a transcription factor, p...

  20. The Usefulness of Selected Physicochemical Indices, Cell Membrane Integrity and Sperm Chromatin Structure in Assessments of Boar Semen Sensitivity

    PubMed Central

    Wysokińska, A.; Kondracki, S.; Iwanina, M.

    2015-01-01

    The present work describes experiments undertaken to evaluate the usefulness of selected physicochemical indices of semen, cell membrane integrity and sperm chromatin structure for the assessment of boar semen sensitivity to processes connected with pre-insemination procedures. The experiments were carried out on 30 boars: including 15 regarded as providers of sensitive semen and 15 regarded as providers of semen that is little sensitive to laboratory processing. The selection of boars for both groups was based on sperm morphology analyses, assuming secondary morphological change incidence in spermatozoa as the criterion. Two ejaculates were manually collected from each boar at an interval of 3 to 4 months. The following analyses were carried out for each ejaculate: sperm motility assessment, sperm pH measurement, sperm morphology assessment, sperm chromatin structure evaluation and cell membrane integrity assessment. The analyses were performed three times. Semen storage did not cause an increase in the incidence of secondary morphological changes in the group of boars considered to provide sperm of low sensitivity. On the other hand, with continued storage there was a marked increase in the incidence of spermatozoa with secondary morphological changes in the group of boars regarded as producing more sensitive semen. Ejaculates of group I boars evaluated directly after collection had an approximately 6% smaller share of spermatozoa with undamaged cell membranes than the ejaculates of boars in group II (p≤0.05). In the process of time the percentage of spermatozoa with undamaged cell membranes decreased. The sperm of group I boars was characterised with a lower sperm motility than the semen of group II boars. After 1 hour of storing diluted semen, the sperm motility of boars producing highly sensitive semen was already 4% lower (p≤0.05), and after 24 hours of storage it was 6.33% lower than that of the boars that produced semen with a low sensitivity. Factors

  1. Use of fluorescently labelled calmodulins as tools to measure subcellular calmodulin activation in living dorsal root ganglion cells.

    PubMed

    Milikan, J M; Bolsover, S R

    2000-01-01

    We have used fluorescently labelled calmodulins to probe the activity of calmodulin in living dorsal root ganglion cells. Calmodulin labelled with the fluorophore 5-([4,6 dichlorotriazin-2yl]amino)-fluorescein (FL-CaM) does not change its fluorescence when it binds calcium, while calmodulin labelled at lysine 75 with 2-chloro-(6-(4-N,N-diethylamino-phenyl)-1,4,5-triazin-4-yl (TA-CaM), an environment-sensitive probe, increases its fluorescence when it binds calcium. We micro-injected FL-CaM or TA-CaM into rat dorsal root ganglion cells and found that both probes localise to the cell nucleus. In contrast, endogenous cellular calmodulin, in dorsal root ganglion cells as in hippocampal neurones, is predominantly cytosolic unless the neurones are depolarised, then it moves to the nucleus. FL-CaM and TA-CaM, introduced into dorsal root ganglion cells via a patch pipette, also immediately move to the nucleus, indicating that the nuclear localisation is a property of the labelled calmodulins. Although the subcellular distribution of FL-CaM and TA-CaM does not necessarily match that of endogenous calmodulin, we show that FL-CaM can be used as a control for TA-CaM when studying calmodulin activation in different cellular compartments.

  2. Noninvasive identification of subcellular organization and nuclear morphology features associated with leukemic cells using light-scattering spectroscopy

    NASA Astrophysics Data System (ADS)

    Hsiao, Austin; Hunter, Martin; Greiner, Cherry; Gupta, Sharad; Georgakoudi, Irene

    2011-03-01

    Leukemia is the most common and deadly cancer among children and one of the most prevalent cancers among adults. Improvements in its diagnosis and monitoring of leukemic patients could have a significant impact in their long-term treatment. We demonstrate that light-scattering spectroscopy (LSS)-based approaches could serve as a tool to achieve this goal. Specifically, we characterize the light scattering properties of leukemic (NALM-6) cells and compare them to those of normal lymphocytes and granulocytes in the 440-710 nm range, over +/-4 deg about the exact backscattering direction. We find that the LSS spectra are well described by an inverse power-law wavelength dependence, with a power exponent insensitive to the scattering angle but significantly higher for leukemic cells than for normal leukocytes. This is consistent with differences in the subcellular morphology of these cells, detected in differential interference contrast images. Furthermore, the residual light-scattering signal, extracted after subtracting the inverse power-law fit from the data, can be analyzed assuming a Gaussian distribution of spherical scatterers using Mie theory. This analysis yields scatterer sizes that are consistent with the diameters of cell nuclei and allows the detection of the larger nuclei of NALM-6 cells compared to those of lymphocytes and granulocytes.

  3. Interaction between the moss Physcomitrella patens and Phytophthora: a novel pathosystem for live-cell imaging of subcellular defence.

    PubMed

    Overdijk, Elysa J R; DE Keijzer, Jeroen; DE Groot, Deborah; Schoina, Charikleia; Bouwmeester, Klaas; Ketelaar, Tijs; Govers, Francine

    2016-08-01

    Live-cell imaging of plant-pathogen interactions is often hampered by the tissue complexity and multicell layered nature of the host. Here, we established a novel pathosystem with the moss Physcomitrella patens as host for Phytophthora. The tip-growing protonema cells of this moss are ideal for visualizing interactions with the pathogen over time using high-resolution microscopy. We tested four Phytophthora species for their ability to infect P. patens and showed that P. sojae and P. palmivora were only rarely capable to infect P. patens. In contrast, P. infestans and P. capsici frequently and successfully penetrated moss protonemal cells, showed intracellular hyphal growth and formed sporangia. Next to these successful invasions, many penetration attempts failed. Here the pathogen was blocked by a barrier of cell wall material deposited in papilla-like structures, a defence response that is common in higher plants. Another common response is the upregulation of defence-related genes upon infection and also in moss we observed this upregulation in tissues infected with Phytophthora. For more advanced analyses of the novel pathosystem we developed a special set-up that allowed live-cell imaging of subcellular defence processes by high-resolution microscopy. With this set-up, we revealed that Phytophthora infection of moss induces repositioning of the nucleus, accumulation of cytoplasm and rearrangement of the actin cytoskeleton, but not of microtubules.

  4. Bovine oviduct epithelial cells downregulate phagocytosis of sperm by neutrophils: prostaglandin E2 as a major physiological regulator.

    PubMed

    Marey, Mohamed A; Liu, Jinghui; Kowsar, Rasoul; Haneda, Shingo; Matsui, Motozumi; Sasaki, Motoki; Takashi, Shimizu; Hayakawa, Hiroyuki; Wijayagunawardane, Missaka P B; Hussein, Fekry M; Miyamoto, Akio

    2014-02-01

    This study aimed to investigate the presence of polymorphonuclear neutrophils (PMNs) in bovine oviduct fluid under physiological conditions and to determine the possible role of bovine oviduct epithelial cells (BOECs) in the regulation of the phagocytic activity of PMNs for sperm. During the pre-ovulatory stage, PMNs were identified in the bovine oviduct fluid in relatively constant numbers. In our experiments, PMNs were incubated for 4 h with the supernatant of cultured BOECs stimulated for 24 h by LH (10 ng/ml). Phagocytosis was then assayed by co-incubation of these PMNs with sperm treated to induce capacitation. The BOEC supernatant significantly suppressed sperm phagocytosis by PMNs, and the LH-stimulated BOEC supernatant further suppressed phagocytosis. Importantly, in the BOEC culture, LH stimulated the secretion of prostaglandin E2 (PGE2), which dose-dependently (10(-6), 10(-7), and 10(-8) M) suppressed sperm phagocytosis by PMNs. Furthermore, a PGEP2 receptor antagonist significantly abrogated the inhibition of phagocytosis by the LH-stimulated BOEC supernatant. Additionally, using scanning electron microscopy, incubation of PMNs with either PGE2 or LH-stimulated BOEC supernatant before phagocytosis was found to prevent the formation of DNA-based neutrophil extracellular traps for sperm entanglement. The results indicate that sperm are exposed to PMNs in the oviduct and PGE2 released into the oviduct fluid after LH stimulation may play a major role in the suppression of the phagocytic activity of PMNs for sperm via interaction with EP2 receptors. Thus, the bovine oviduct provides a PGE2-rich microenvironment to protect sperm from phagocytosis by PMNs, thereby supporting sperm survival in the oviduct. Free Japanese abstract A Japanese translation of this abstract is freely available at http://www.reproduction-online.org/content/147/2/211/suppl/DC1.

  5. Missense mutations in Otopetrin 1 affect subcellular localization and inhibition of purinergic signaling in vestibular supporting cells.

    PubMed

    Kim, Euysoo; Hyrc, Krzysztof L; Speck, Judith; Salles, Felipe T; Lundberg, Yunxia W; Goldberg, Mark P; Kachar, Bechara; Warchol, Mark E; Ornitz, David M

    2011-03-01

    Otopetrin 1 (Otop1) encodes a protein that is essential for the development of otoconia. Otoconia are the extracellular calcium carbonate containing crystals that are important for vestibular mechanosensory transduction of linear motion and gravity. There are two mutant alleles of Otop1 in mice, titled (tlt) and mergulhador (mlh), which result in non-syndromic otoconia agenesis and a consequent balance defect. Biochemically, Otop1 has been shown to modulate purinergic control of intracellular calcium in vestibular supporting cells, which could be one of the mechanisms by which Otop1 participates in the mineralization of otoconia. To understand how tlt and mlh mutations affect the biochemical function of Otop1, we examined the purinergic response of COS7 cells expressing mutant Otop1 proteins, and dissociated sensory epithelial cells from tlt and mlh mice. We also examined the subcellular localization of Otop1 in whole sensory epithelia from tlt and mlh mice. Here we show that tlt and mlh mutations uncouple Otop1 from inhibition of P2Y receptor function. Although the in vitro biochemical function of the Otop1 mutant proteins is normal, in vivo they behave as null alleles. We show that in supporting cells the apical membrane localization of the mutant Otop1 proteins is lost. These data suggest that the tlt and mlh mutations primarily affect the localization of Otop1, which interferes with its ability to interact with other proteins that are important for its cellular and biochemical function.

  6. Galactosylceramidase deficiency causes sperm abnormalities in the mouse model of globoid cell leukodystrophy

    SciTech Connect

    Luddi, A.; Strazza, M.; Carbone, M.; Moretti, E.; Costantino-Ceccarini, E. . E-mail: costantino@unisi.it

    2005-03-10

    The classical recessive mouse mutant, 'the twitcher,' is one of the several animal models of the human globoid cell leukodystrophy (Krabbe disease) caused by a deficiency in the gene encoding the lysosomal enzyme galactosylceramidase (GALC). The failure to hydrolyze galactosylceramide (gal-cer) and galactosylsphingosine (psychosine) leads to degeneration of oligodendrocytes and severe demyelination. Substrate for GALC is also the galactosyl-alkyl-acyl-glycerol (GalAAG), precursor of the seminolipid, the most abundant glycolipid in spermatozoa of mammals. In this paper, we report the pathobiology of the testis and sperm in the twitcher mouse and demonstrate the importance of GALC for normal sperm maturation and function. The GALC deficit results in accumulation of GalAAG in the testis of the twitcher mouse. Morphological studies revealed that affected spermatozoa have abnormally swollen acrosomes and angulation of the flagellum mainly at midpiece-principal piece junction. Multiple folding of the principal piece was also observed. Electron microscopy analysis showed that in the twitcher sperm, acrosomal membrane is redundant, detached from the nucleus and folded over. Disorganization and abnormal arrangements of the axoneme components were also detected. These results provide in vivo evidence that GALC plays a critical role in spermiogenesis.

  7. Real-time Imaging and Tuning Subcellular Structures and Membrane Transport Kinetics of Single Live Cells at Nanosecond Regime

    PubMed Central

    Xu, Hongwu; Nallathamby, Prakash D.; Xu, Xiao-Hong Nancy

    2009-01-01

    We developed an electric-field exposure microchannel system with 230-nanometer thin-layer gold electrodes, and interfaced it with a single living cell imaging station and a 10-nanosecond-electric-pulse (10nsEP) generator. This design allows us to image intracellular molecules and structures, membrane transport and viability of single leukemic cells (HL60) while the cells are exposed to 10nsEPs of 0–179 kV/cm, permitting the study of subcellular responses at nanosecond regime. The electrodes confine a thin-layer section of the cells exposed to 10nsEPs, offering unprecedented high spatial resolution (230-nm at z-direction of E and imaging plane) for imaging intracellular molecules of single cells affected by 10nsEPs. We found that nucleic acids, membrane transport rates and viability of single cells depend on the number and electric-field-strength (E) of 10nsEPs, showing the cumulative effect of 10nsEPs on intracellular molecules and structures and suggesting the possibility of tuning them one-pulse-at-a-time. Using lower E (51 kV/cm) of 10nsEPs, we could manipulate nucleic acids of single living cells without disrupting their cellular membrane and viability. As E increases to 80, 124 and 179 kV/cm, membrane integrity and viability of cells exhibit higher dependence on the number of 10nsEPs in a non-linear fashion, showing that critical E and pulse number are needed to surmount cellular transport barriers and membrane integrity. PMID:19795898

  8. Effects of cinnamon (Cinnamomum zeylanicum) bark oil on testicular antioxidant values, apoptotic germ cell and sperm quality.

    PubMed

    Yüce, A; Türk, G; Çeribaşi, S; Sönmez, M; Çiftçi, M; Güvenç, M

    2013-08-01

    Cinnamon and its contents have multifactorial properties such as antioxidant, anti-inflammatory and antidiabetic. Male infertility is one of the major health problems in life. The aim of this study was to investigate the effects of long-term cinnamon bark oil (CBO) ingestion on testicular antioxidant values, apoptotic germ cell and sperm quality of adult rats. Twelve male healthy Wistar rats were divided into two groups, each group containing six rats. While olive oil was given to control group, 100 mg kg(-1)  CBO was administered to the other group by gavage daily for 10 weeks. Body and reproductive organ weights, sperm characteristics, testicular lipid peroxidation and antioxidant enzyme activities, and testicular apoptosis via terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) method were examined. A significant decrease in malondialdehyde level and marked increases in reduced glutathione level, glutathione peroxidase and catalase activities were observed in rats treated with CBO compared with the control group. CBO consumption provided a significant increase in weights of testes and epididymides, epididymal sperm concentration, sperm motility and diameter of seminiferous tubules when compared with the control group. However, CBO consumption tended to decrease the abnormal sperm rate and apoptotic germ cell count, but it did not reach statistical significance. It is concluded that CBO has improvement effect on testicular oxidant-antioxidant balance and sperm quality, and its consumption may be useful for asthenozoospermic men.

  9. Seminal cell-free DNA levels measured by PicoGreen fluorochrome are associated with sperm fertility criteria.

    PubMed

    Costa, F; Barbisan, F; Assmann, C E; Araújo, N K F; de Oliveira, A R; Signori, J P; Rogalski, F; Bonadiman, B; Fernandes, M S; da Cruz, I B M

    2017-03-07

    Previous investigations suggested that elevated cell-free DNA (cfDNA) can indicate non-healthy states. However, the potential association between cfDNA seminal plasma levels and fertility sperm parameters has not yet been determined. Therefore, the present study evaluated the association between seminal cfDNA levels and sperm fertility criteria to determine the use of seminal cfDNA quantification. An in vivo protocol quantified cfDNA levels of semen samples obtained from 163 male patients using fluorescent PicoGreen dye staining. To confirm if semen cfDNA quantification is realistic, an in vitro complementary test was performed using three or four semen samples. The fresh sperm samples were exposed to paraquat that generates high levels of superoxide anion causing oxidative stress and cell mortality. The results showed significant association between dsDNA levels and several sperm fertility parameters, such as low viability and alterations of motility and morphology. The in vitro analysis confirmed the association between dsDNA levels and sperm viability. Together, these results suggest that dsDNA levels could be an important biomarker to test sperm fertility.

  10. Subcellular compartment-specific molecular diversity of pre- and postsynaptic GABAB-activated GIRK channels in Purkinje cells

    PubMed Central

    Fernández-Alacid, Laura; Aguado, Carolina; Ciruela, Francisco; Martín, Ricardo; Colón, José; Cabañero, María José; Gassmann, Martin; Watanabe, Masahiko; Shigemoto, Ryuichi; Wickman, Kevin; Bettler, Bernhard; Sánchez-Prieto, José; Luján, Rafael

    2009-01-01

    Activation of G protein-gated inwardly-rectifying K+ (GIRK or Kir3) channels by metabotropic gamma-aminobutyric acid (B) (GABAB) receptors is an essential signalling pathway controlling neuronal excitability and synaptic transmission in the brain. To investigate the relationship between GIRK channel subunits and GABAB receptors in cerebellar Purkinje cells at post- and pre-synaptic sites, we used biochemical, functional and immunohistochemical techniques. Co-immunoprecipitation analysis demonstrated that GIRK subunits are co-assembled with GABAB receptors in the cerebellum. Immunoelectron microscopy showed that the subunit composition of GIRK channels in Purkinje cell spines is compartment-dependent. Thus, at extrasynaptic sites GIRK channels are formed by GIRK1/GIRK2/GIRK3, postsynaptic densities contain GIRK2/GIRK3 and dendritic shafts contain GIRK1/GIRK3. The postsynaptic association of GIRK subunits with GABAB receptors in Purkinje cells is supported by the subcellular regulation of the ion channel and the receptor in mutant mice. At presynaptic sites, GIRK channels localized to parallel fibre terminals are formed by GIRK1/GIRK2/GIRK3 and co-localize with GABAB receptors. Consistent with this morphological evidence we demonstrate their functional interaction at axon terminals in the cerebellum by showing that GIRK channels play a role in the inhibition of glutamate release by GABAB receptors. The association of GIRK channels and GABAB receptors with excitatory synapses at both post- and presynaptic sites indicates their intimate involvement in the modulation of glutamatergic neurotransmission in the cerebellum. PMID:19558451

  11. Cell population indexes of spermatogenic yield and testicular sperm reserves in adult jaguars (Panthera onca).

    PubMed

    de Azevedo, Maria Helena Ferreira; de Paula, Tarcízio Antônio Rego; Matta, Sérgio Luis Pinto da; Fonseca, Cláudio César; da Costa, Eduardo Paulino; Costa, Deiler Sampaio; Peixoto, Juliano Vogas

    2010-03-01

    The intrinsic yield of spermatogenesis and supporting capacity of Sertoli cells are the desirable indicators of sperm production in a species. The objective of the present study was to quantify intrinsic yield and the Sertoli cell index in the spermatogenic process and estimate testicular sperm reserves by histological assessment of fragments obtained by testicular biopsy of five adult jaguars in captivity. The testicular fragments were fixed in 4% glutaric aldehyde, dehydrated at increasing alcohol concentrations, included into hydroxyethyl methacrylate, and were cut into 4 microm thickness. In the seminiferous epithelium of the jaguar, 9.2 primary spermatocytes in pre-leptotene were produced by "A" spermatogonia. During the meiotic divisions only 3.2 spermatids were produced by a primary spermatocyte. The general spermatogenic yield of the jaguar was about 23.4 cells and each Sertoli cell was able to maintain about 19.2 germ cells, 11 of them were round spermatids. In each seminiferous epithelium cycle about 166 million spermatozoa were produced by each gram of testicular tissue. In adult jaguars, the general spermatogenic yield was similar to the yield observed in pumas, greater than that observed for the domestic cat, but less compared to most domestic animals.

  12. Mammalian sperm morphometry.

    PubMed Central

    Gage, M J

    1998-01-01

    Understanding the adaptive significance of sperm form and function has been a challenge to biologists because sperm are highly specialized cells operating at a microscopic level in a complex environment. A fruitful course of investigation has been to use the comparative approach. This comparative study attempts to address some fundamental questions of the evolution of mammalian sperm morphometry. Data on sperm morphometry for 445 mammalian species were collated from published sources. I use contemporary phylogenetic analysis to control for the inherent non-independence of species and explore relationships between the morphometric dimensions of the three essential spermatozoal components: head, mid-piece and flagellum. Energy for flagellar action is metabolized by the mitochondrial-dense mid-piece and these combine to propel the sperm head, carrying the male haplotype, to the ovum. I therefore search for evolutionary associations between sperm morphometry and body mass, karyotype and the duration of oestrus. In contrast to previous findings, there is no inverse correlation between body weight and sperm length. Sperm mid-piece and flagellum lengths are positively associated with both head length and area, and the slopes of these relationships are discussed. Flagellum length is positively associated with mid-piece length but, in contrast to previous research and after phylogenetic control, I find no relationship between flagellum length and the volume of the mitochondrial sheath. Sperm head dimensions are not related to either genome mass or chromosome number, and there are no relationships between sperm morphometry and the duration of oestrus. PMID:9474794

  13. Effect of NGF on the subcellular localization of group IIA secretory phospholipase A(2) (GIIA) in PC12 cells: role in neuritogenesis.

    PubMed

    Ferrini, M; Nardicchi, V; Mannucci, R; Arcuri, C; Nicoletti, I; Donato, R; Goracci, G

    2010-12-01

    Phospholipases A(2) (PLA(2)s) are involved in neuritogenesis but the identity of the isoforms(s) contributing to this process is still not defined. Several reports have focused on secretory PLA(2)s (sPLA(2)) as the administration of exogenous sPLA(2)s to PC12 neuronal cells stimulates neurite outgrowth. The present study demonstrates that the endogenous group IIA sPLA(2) (GIIA), constitutively expressed in mammalian neural cells, changes its subcellular localization when PC12 cells are induced to differentiate by NGF treatment. Indeed, confocal analysis showed a time-dependent accumulation of GIIA in growth cones and neurite tips. Under identical conditions the subcellular distribution of another isoform (GV) was unaffected by NGF. Contrary to GX, another sPLA(2) isoform expressed by PC12 cells, the contribution of GIIA to neuritogenesis does not require its release in the extracellular medium.

  14. Characterization of secretory proteins from cultured cauda epididymal cells that significantly sustain bovine sperm motility in vitro.

    PubMed

    Reyes-Moreno, Carlos; Boilard, Mathieu; Sullivan, Robert; Sirard, Marc-André

    2002-12-01

    Epididymis provides a safe environment in which stored-spermatozoa could survive for days before ejaculation. In vitro studies suggested that epididymal proteins seem to be implicated in sperm survival during coincubation with cultured epididymal cells. This study was basically designed to confirm if secretory proteins from bovine epididymal cell cultures provide sperm protection against rapid loss of sperm motility in vitro. Bovine spermatozoa were incubated in conditioned media (CM), which were prepared from cultured cauda epididymal cell (CEC). Motion parameters were recorded using a computer-assisted sperm analyzer. Sperm-free protein extracts from CM were fractionated by ultrafiltration through a 10-kDa cut off membrane. A significantly positive effect on sperm motility was observed when spermatozoa were incubated in CM (54 +/- 4%) and CM > 10 kDa (57 +/- 4%) compared to CM < 10-kDa fraction (30 +/- 3%) or fresh media (34 +/- 3%), after a 6-hr incubation period. This beneficial effect on sperm motility was abolished when the CM > 10-kDa fraction was heat-treated at 100 degrees C for 10 min. The CM > 10 kDa fraction provides factors that remained active even though spermatozoa were washed twice after a 2-hr preincubation period. To identify potential beneficial factors, bovine spermatozoa were incubated with radiolabeled proteins obtained using (35)S-methionine in culture medium. SDS-PAGE analysis of proteins extracted from CM-preincubated spermatozoa revealed the presence of a 42-kDa protein strongly associated to the sperm surface. This 42-kDa spot was trypsin-digested and identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as a protein homologue to a 35-kDa bovine estrogen-sulfotransferase. This protein can play a role in epididymal biology and sperm function. Taken together, these results suggest that specific epididymal proteins can be implicated in the sperm protection in vitro, and can be characterized in our cell culture

  15. The Role of Lipid Droplets in Mortierella alpina Aging Revealed by Integrative Subcellular and Whole-Cell Proteome Analysis.

    PubMed

    Yu, Yadong; Li, Tao; Wu, Na; Jiang, Ling; Ji, Xiaojun; Huang, He

    2017-03-07

    Lipid droplets (LDs) participate in many cellular processes in oleaginous microorganisms. However, the exact function of LDs in the Mortierella alpina aging process remains elusive. Herein, subcellular proteomics was employed to unveil the composition and dynamics of the LD proteome in the aging M. alpina for the first time. More than 400 proteins were detected in LDs and 62 of them changed expression significantly during aging. By combining the LD proteomic data with whole-cell data, we found that the carbohydrate metabolism and de novo lipid biosynthesis were all inhibited during aging of M. alpina mycelia. The up-regulation of fructose metabolism-related enzymes in LDs might imply that LDs facilitated the fructose metabolism, which in turn might cause pyruvate to accumulate and enter malate-pyruvate cycle, and ultimately, provide additional NADPH for the synthesis of arachidonic acid (ARA). Lysophospholipase and lecithinase were up-regulated in LDs during the aging process, suggesting that the phospholipids and lecithin were starting to be hydrolyzed, in order to release fatty acids for the cells. The impairment of the anti-oxidant system might lead to the accumulation of ROS and consequently cause the up-regulation of autophagy-related proteins in LDs, which further induces the M. alpina mycelia to activate the autophagy process.

  16. The Role of Lipid Droplets in Mortierella alpina Aging Revealed by Integrative Subcellular and Whole-Cell Proteome Analysis

    PubMed Central

    Yu, Yadong; Li, Tao; Wu, Na; Jiang, Ling; Ji, Xiaojun; Huang, He

    2017-01-01

    Lipid droplets (LDs) participate in many cellular processes in oleaginous microorganisms. However, the exact function of LDs in the Mortierella alpina aging process remains elusive. Herein, subcellular proteomics was employed to unveil the composition and dynamics of the LD proteome in the aging M. alpina for the first time. More than 400 proteins were detected in LDs and 62 of them changed expression significantly during aging. By combining the LD proteomic data with whole-cell data, we found that the carbohydrate metabolism and de novo lipid biosynthesis were all inhibited during aging of M. alpina mycelia. The up-regulation of fructose metabolism-related enzymes in LDs might imply that LDs facilitated the fructose metabolism, which in turn might cause pyruvate to accumulate and enter malate-pyruvate cycle, and ultimately, provide additional NADPH for the synthesis of arachidonic acid (ARA). Lysophospholipase and lecithinase were up-regulated in LDs during the aging process, suggesting that the phospholipids and lecithin were starting to be hydrolyzed, in order to release fatty acids for the cells. The impairment of the anti-oxidant system might lead to the accumulation of ROS and consequently cause the up-regulation of autophagy-related proteins in LDs, which further induces the M. alpina mycelia to activate the autophagy process. PMID:28266581

  17. Impact of Bep or Carboplatin Chemotherapy on Testicular Function and Sperm Nucleus of Subjects with Testicular Germ Cell Tumor

    PubMed Central

    Ghezzi, Marco; Berretta, Massimiliano; Bottacin, Alberto; Palego, Pierfrancesco; Sartini, Barbara; Cosci, Ilaria; Finos, Livio; Selice, Riccardo; Foresta, Carlo; Garolla, Andrea

    2016-01-01

    Young males have testicular germ cells tumors (TGCT) as the most common malignancy and its incidence is increasing in several countries. Besides unilateral orchiectomy (UO), the treatment of TGCT may include surveillance, radiotherapy, or chemotherapy (CT), basing on tumor histology and stage of disease. It is well known that both radio and CT may have negative effects on testicular function, affecting spermatogenesis, and sex hormones. Many reports investigated these aspects in patients treated with bleomycin, etoposide, and cisplatin (BEP), after UO. In contrast no data are available on the side effects of carboplatin treatment in these patients. We included in this study 212 consecutive subjects who undergone to sperm banking at our Andrology and Human Reproduction Unit after UO for TGCT. Hundred subjects were further treated with one or more BEP cycles (BEP-group), 54 with carboplatin (CARB group), and 58 were just surveilled (S-group). All patients were evaluated for seminal parameters, sperm aneuploidy, sperm DNA, sex hormones, volume of the residual testis at baseline (T0) and after 12 (T1) and 24 months (T2) from UO or end of CT. Seminal parameters, sperm aneuploidies, DNA status, gonadic hormones, and testicular volume at baseline were not different between groups. At T1, we observed a significant reduction of sperm concentration and sperm count in the BEP group versus baseline and versus both Carb and S-group. A significant increase of sperm aneuploidies was present at T1 in the BEP group. Similarly, the same group at 1 had altered sperm DNA integrity and fragmentation compared with baseline, S-group and Carb group. These alterations were persistent after 2 years from the end of BEP treatment. Despite a slight improvement at T2, the BEP group had still higher percentages of sperm aneuploidies than other groups. No impairment of sperm aneuploidies and DNA status were observed in the Carb group both after 1 and 2 years from the end of treatment. Despite

  18. Direct contact between boar spermatozoa and porcine oviductal epithelial cell (OEC) cultures is needed for optimal sperm survival in vitro.

    PubMed

    Yeste, M; Lloyd, R E; Badia, E; Briz, M; Bonet, S; Holt, W V

    2009-07-01

    Oviductal epithelial cell (OEC) co-culture prolongs sperm viability and motility in vitro in a number of species including humans and horses. This study has sought to determine the effects of homologous OEC co-culture on boar sperm function. To determine whether the effects on spermatozoa were specifically caused by co-culture with or by OEC secretions, or by both factors together, a number of co-culture and cell-conditioned medium (CM) experiments were conducted. Firstly, Percoll-washed spermatozoa were co-cultured with OECs and pig kidney epithelial (LLC-PK1) cells, and in medium without cells. Secondly, Percoll-washed spermatozoa were incubated with CM derived from both OECs and LLC-PK1 cells and in unconditioned medium. A number of sperm function parameters were assessed after 5, 30, 60, 90, 120, and 180 min, and 24h of co-culturing or incubation with CM. Of all the sperm function parameters investigated, the percentage (%) viability data yielded the most interesting results. OECs (mean+/-S.E.M.; 31.2+/-1.10) were better than LLC-PK1 cells (24.3+/-0.93) at prolonging the viability of unbound spermatozoa after 24h of co-culturing (P<0.05). Also after 24h, the viability of spermatozoa bound to the OECs (77.6+/-1.83) was significantly higher than in the case of the LLC-PK1 cells (53.5+/-1.43; P<0.001). Other sperm function parameters, e.g., capacitation and motility, were also influenced by OEC co-culturing and incubation with CM, although to a lesser degree. In conclusion, porcine homologous OEC co-culture and CM incubation specifically affect sperm function. However, we propose that it is OEC co-culturing, rather than OEC-CM, that has the greater influence.

  19. Proteomic Analyses Reveal Common Promiscuous Patterns of Cell Surface Proteins on Human Embryonic Stem Cells and Sperms

    PubMed Central

    Gu, Bin; Zhang, Jiarong; Wu, Ying; Zhang, Xinzong; Tan, Zhou; Lin, Yuanji; Huang, Xiao; Chen, Liangbiao; Yao, Kangshou; Zhang, Ming

    2011-01-01

    Background It has long been proposed that early embryos and reproductive organs exhibit similar gene expression profiles. However, whether this similarity is propagated to the protein level remains largely unknown. We have previously characterised the promiscuous expression pattern of cell surface proteins on mouse embryonic stem (mES) cells. As cell surface proteins also play critical functions in human embryonic stem (hES) cells and germ cells, it is important to reveal whether a promiscuous pattern of cell surface proteins also exists for these cells. Methods and Principal Findings Surface proteins of hES cells and human mature sperms (hSperms) were purified by biotin labelling and subjected to proteomic analyses. More than 1000 transmembrane or secreted cell surface proteins were identified on the two cell types, respectively. Proteins from both cell types covered a large variety of functional categories including signal transduction, adhesion and transporting. Moreover, both cell types promiscuously expressed a wide variety of tissue specific surface proteins, and some surface proteins were heterogeneously expressed. Conclusions/Significance Our findings indicate that the promiscuous expression of functional and tissue specific cell surface proteins may be a common pattern in embryonic stem cells and germ cells. The conservation of gene expression patterns between early embryonic cells and reproductive cells is propagated to the protein level. These results have deep implications for the cell surface signature characterisation of pluripotent stem cells and germ cells and may lead the way to a new area of study, i.e., the functional significance of promiscuous gene expression in pluripotent and germ cells. PMID:21559292

  20. Using Femtosecond Laser Subcellular Surgery as a Tool to Study Cell Biology

    SciTech Connect

    Shen, N; Colvin, M E; Huser, T

    2007-02-27

    Research on cellular function and regulation would be greatly advanced by new instrumentation using methods to alter cellular processes with spatial discrimination on the nanometer-scale. We present a novel technique for targeting submicrometer sized organelles or other biologically important regions in living cells using femtosecond laser pulses. By tightly focusing these pulses beneath the cell membrane, we can vaporize cellular material inside the cell through nonlinear optical processes. This technique enables non-invasive manipulation of the physical structure of a cell with sub-micrometer resolution. We propose to study the role mitochondria play in cell proliferation and apoptosis. Our technique provides a unique tool for the study of cell biology.

  1. Single-cell and subcellular pharmacokinetic imaging allows insight into drug action in vivo

    PubMed Central

    Thurber, Greg M.; Yang, Katy S.; Reiner, Thomas; Kohler, Rainer H.; Sorger, Peter; Mitchison, Tim; Weissleder, Ralph

    2013-01-01

    Pharmacokinetic analysis at the organ level provides insight into how drugs distribute throughout the body but cannot explain how drugs work at the cellular level. Here we demonstrate in vivo single cell pharmacokinetic imaging of PARP-1 inhibitors (PARPi) and model drug behavior under varying conditions. We visualize intracellular kinetics of PARPi distribution in real time, showing that PARPi reaches its cellular target compartment, the nucleus, within minutes in vivo both in cancer and normal cells in various cancer models. We also use these data to validate predictive finite element modeling. Our theoretical and experimental data indicate that tumor cells are exposed to sufficiently high PARPi concentrations in vivo and suggest that drug inefficiency is likely related to proteomic heterogeneity or insensitivity of cancer cells to DNA repair inhibition. This suggests that single cell pharmacokinetic imaging and derived modeling improves our understanding of drug action at single cell resolution in vivo. PMID:23422672

  2. Tyrosine phosphorylation within the SH3 domain regulates CAS subcellular localization, cell migration, and invasiveness.

    PubMed

    Janoštiak, Radoslav; Tolde, Ondřej; Brůhová, Zuzana; Novotný, Marian; Hanks, Steven K; Rösel, Daniel; Brábek, Jan

    2011-11-01

    Crk-associated substrate (CAS) is a major tyrosine-phosphorylated protein in cells transformed by v-crk and v-src oncogenes and plays an important role in invasiveness of Src-transformed cells. A novel phosphorylation site on CAS, Tyr-12 (Y12) within the ligand-binding hydrophobic pocket of the CAS SH3 domain, was identified and found to be enriched in Src-transformed cells and invasive human carcinoma cells. To study the biological significance of CAS Y12 phosphorylation, phosphomimicking Y12E and nonphosphorylatable Y12F mutants of CAS were studied. The phosphomimicking mutation decreased interaction of the CAS SH3 domain with focal adhesion kinase (FAK) and PTP-PEST and reduced tyrosine phosphorylation of FAK. Live-cell imaging showed that green fluorescent protein-tagged CAS Y12E mutant is, in contrast to wild-type or Y12F CAS, excluded from focal adhesions but retains its localization to podosome-type adhesions. Expression of CAS-Y12F in cas-/- mouse embryonic fibroblasts resulted in hyperphosphorylation of the CAS substrate domain, and this was associated with slower turnover of focal adhesions and decreased cell migration. Moreover, expression of CAS Y12F in Src-transformed cells greatly decreased invasiveness when compared to wild-type CAS expression. These findings reveal an important role of CAS Y12 phosphorylation in the regulation of focal adhesion assembly, cell migration, and invasiveness of Src-transformed cells.

  3. Abnormal sperm morphology in mouse germ cells after short-term exposures to acetamiprid, propineb, and their mixture.

    PubMed

    Rasgele, Pinar Göç

    2014-03-01

    Pesticides are one of the most potent environmental contaminants, which accumulate in biotic and abiotic components of ecosystems. Acetamiprid (Acm), a neonicotinoid insecticide, and Propineb (Pro), a dithiocarbamate fungicide, are widely used to control sucking insects and fungal infections on crops, respectively. The present study was undertaken to investigate the genotoxic effects of these compounds, individually and in mixtures, in mouse germ cells by using the sperm morphology assay. Mice were injected intraperitoneally with 0.625, 1.25, and 2.50 μg mL⁻¹ of Acm, 12.5, 25, and 50 μg mL⁻¹ of Pro, and their mixture at the same concentrations over 24 and 48 h. Acm did not significantly increase the percentage of abnormal sperm at any concentration. The frequency of abnormal sperm significantly increased after 24 and 48 h of exposure to 50 μg mL⁻¹ of Pro. The mixtures of 2.50 μg mL⁻¹ of Acm and 50 μg mL⁻¹ of Pro induced sperm abnormalities antagonistically both after 24 and 48 h of exposure. Results suggest that Acm was non-genotoxic for mouse germ cells, while Pro may have been a germ cell mutagen due to the observed increase in the frequency of sperm abnormalities. However, to gain better insight into the mutagenicity and DNA damaging potential of both of these pesticides, further studies at molecular level should be done.

  4. Analysis of Nuclear RNA Interference (RNAi) in Human Cells by Subcellular Fractionation and Argonaute Loading

    PubMed Central

    Gagnon, Keith T.; Li, Liande; Janowski, Bethany A.; Corey, David R.

    2014-01-01

    RNA interference (RNAi) is well known for its ability to regulate gene expression in the cytoplasm of mammalian cells. In mammalian cell nuclei, however, the impact of RNAi has remained more controversial. A key technical hurdle has been a lack of optimized protocols for the isolation and analysis of cell nuclei. Here we describe a simplified protocol for nuclei isolation from cultured cells that incorporates a method for obtaining nucleoplasmic and chromatin fractions and removing cytoplasmic contamination. Cell fractions can then be used to detect the presence and activity of RNAi factors in the nucleus. We present a protocol for investigating an early step in RNAi, Argonaute protein loading with small RNAs, which is enabled by our improved extract preparations. These protocols facilitate characterization of nuclear RNAi and can be applied to the analysis of other nuclear proteins and pathways. From cellular fractionation to analysis of Argonaute loading results, this protocol takes 4–6 d to complete. PMID:25079428

  5. Thin-film optoacoustic transducers for subcellular Brillouin oscillation imaging of individual biological cells.

    PubMed

    Pérez-Cota, Fernando; Smith, Richard J; Moradi, Emilia; Marques, Leonel; Webb, Kevin F; Clark, Matt

    2015-10-01

    At low frequencies ultrasound is a valuable tool to mechanically characterize and image biological tissues. There is much interest in using high-frequency ultrasound to investigate single cells. Mechanical characterization of vegetal and biological cells by measurement of Brillouin oscillations has been demonstrated using ultrasound in the GHz range. This paper presents a method to extend this technique from the previously reported single-point measurements and line scans into a high-resolution acoustic imaging tool. Our technique uses a three-layered metal-dielectric-metal film as a transducer to launch acoustic waves into the cell we want to study. The design of this transducer and measuring system is optimized to overcome the vulnerability of a cell to the exposure of laser light and heat without sacrificing the signal-to-noise ratio. The transducer substrate shields the cell from the laser radiation, efficiently generates acoustic waves, facilitates optical detection in transmission, and aids with heat dissipation away from the cell. This paper discusses the design of the transducers and instrumentation and presents Brillouin frequency images on phantom, fixed, and living cells.

  6. Subcellular localization of adenosine kinase in mammalian cells: The long isoform of AdK is localized in the nucleus

    SciTech Connect

    Cui, Xianying Amy; Singh, Bhag; Park, Jae; Gupta, Radhey S.

    2009-10-09

    Two isoforms of adenosine kinase (AdK) have been identified in mammalian organisms with the long isoform (AdK-long) containing extra 20-21 amino acids at the N-terminus (NTS). The subcellular localizations of these isoforms are not known and they contain no identifiable targeting sequence. Immunofluorescence labeling of mammalian cells expressing either only AdK-long or both isoforms with AdK-specific antibody showed only nuclear labeling or both nucleus and cytoplasmic labeling, respectively. The AdK-long and -short isoforms fused at the C-terminus with c-myc epitope also localized in the nucleus and cytoplasm, respectively. Fusion of the AdK-long NTS to green fluorescent protein also resulted in its nuclear localization. AdK-long NTS contains a cluster of conserved amino acids (PKPKKLKVE). Replacement of KK in this sequence with either AA or AD abolished its nuclear localization capability, indicating that this cluster likely serves as a nuclear localization signal. AdK in nucleus is likely required for sustaining methylation reactions.

  7. Subcellular Localization of Dhurrin β-Glucosidase and Hydroxynitrile Lyase in the Mesophyll Cells of Sorghum Leaf Blades 1

    PubMed Central

    Thayer, Susan S.; Conn, Eric E.

    1981-01-01

    Studies with purified mesophyll and epidermal protoplasts and bundle sheath strands have shown that the cyanogenic glucoside dhurrin (p-hydroxy-(S)-mandelonitrile-β-d-glucoside) is localized in the epidermis of sorghum leaves whereas the enzymes involved in its degradation (dhurrin β-glucosidase and hydroxynitrile lyase) are localized in the mesophyll tissue (Kojima M, JE Poulton, SS Thayer, EE Conn 1979 Plant Physiol 63: 1022-1028). The subcellular localization of these enzymes has now been examined using linear 30 to 55% (w/w) sucrose gradients by fractionation of mesophyll protoplast components. The hydroxynitrile lyase is found in the supernatant fractions suggesting a cytoplasmic (soluble cytoplasm, microsomal or vacuolar location). The dhurrin β-glucosidase (dhurrinase) is particulate and mostly chloroplast-associated. The dhurrinase activity peak has a shoulder of activity more dense than that of the intact chloroplasts. This shoulder does not coincide with markers of any other cell fraction. In studies of chloroplasts isolated from ruptured mesophyll protoplasts by differential, low-speed centrifugation, the dhurrinase partitions in the same manner as the chloroplast marker triose phosphate dehydrogenase. Chloroplast localization of the β-glucosidase has also been shown in histochemical studies using 6-bromo-2-naphthyl-β-d-glucoside substrate coupled with fast Blue B. Images PMID:16661725

  8. Expression and subcellular targeting of canine parvovirus capsid proteins in baculovirus-transduced NLFK cells.

    PubMed

    Gilbert, Leona; Välilehto, Outi; Kirjavainen, Sanna; Tikka, Päivi J; Mellett, Mark; Käpylä, Pirjo; Oker-Blom, Christian; Vuento, Matti

    2005-01-17

    A mammalian baculovirus delivery system was developed to study targeting in Norden Laboratories feline kidney (NLFK) cells of the capsid proteins of canine parvovirus (CPV), VP1 and VP2, or corresponding counterparts fused to EGFP. VP1 and VP2, when expressed alone, both had equal nuclear and cytoplasmic distribution. However, assembled form of VP2 had a predominantly cytoplasmic localization. When VP1 and VP2 were simultaneously present in cells, their nuclear localization increased. Thus, confocal immunofluorescence analysis of cells transduced with the different baculovirus constructs or combinations thereof in the absence or presence of infecting CPV revealed that the VP1 protein is a prerequisite for efficient targeting of VP2 to the nucleus. The baculovirus vectors were functional and the genes of interest efficiently introduced to this CPV susceptible mammalian cell line. Thus, we show evidence that the system could be utilized to study targeting of the CPV capsid proteins.

  9. Cell-permeable peptide-based disruption of endogenous PKA-AKAP complexes: a tool for studying the molecular roles of AKAP-mediated PKA subcellular anchoring.

    PubMed

    Faruque, Omar M; Le-Nguyen, Dung; Lajoix, Anne-Dominique; Vives, Eric; Petit, Pierre; Bataille, Dominique; Hani, El-Habib

    2009-02-01

    Stimulation of numerous G protein-coupled receptors leads to the elevation of intracellular concentrations of cAMP, which subsequently activates the PKA pathway. Specificity of the PKA signaling module is determined by a sophisticated subcellular targeting network that directs the spatiotemporal activation of the kinase. This specific compartmentalization mechanism occurs through high-affinity interactions of PKA with A-kinase anchoring proteins (AKAPs), the role of which is to target the kinase to discrete subcellular microdomains. Recently, a peptide designated "AKAPis" has been proposed to competitively inhibit PKA-AKAP interactions in vitro. We therefore sought to characterize a cell-permeable construct of the AKAPis inhibitor and use it as a tool to characterize the impact of PKA compartmentalization by AKAPs. Using insulin-secreting pancreatic beta-cells (INS-1 cells), we showed that TAT-AKAPis (at a micromolar range) dose dependently disrupted a significant fraction of endogenous PKA-AKAP interactions. Immunoflurescent analysis also indicated that TAT-AKAPis significantly affected PKA subcellular localization. Furthermore, TAT-AKAPis markedly attenuated glucagon-induced phosphorylations of p44/p42 MAPKs and cAMP response element binding protein, which are downstream effectors of PKA. In parallel, TAT-AKAPis dose dependently inhibited the glucagon-induced potentiation of insulin release. Therefore, AKAP-mediated subcellular compartmentalization of PKA represents a key mechanism for PKA-dependent phosphorylation events and potentiation of insulin secretion in intact pancreatic beta-cells. More interestingly, our data highlight the effectiveness of the cell-permeable peptide-mediated approach to monitoring in cellulo PKA-AKAP interactions and delineating PKA-dependent phosphorylation events underlying specific cellular responses.

  10. FOXP3 Subcellular Localization Predicts Recurrence in Oral Squamous Cell Carcinoma

    PubMed Central

    Weed, Donald T.; Walker, Gail; De La Fuente, Adriana C.; Nazarian, Ronen; Vella, Jennifer L.; Gomez-Fernandez, Carmen R.; Serafini, Paolo

    2013-01-01

    Forkhead box protein P3 (FOXP3) expression in tumor infiltrating CD4+T cells is generally associated with an intrinsic capacity to suppress tumor immunity. Based on this notion, different studies have evaluated the prognostic value of this maker in cancer but contradictory results have been found. Indeed, even within the same cancer population, the presence of CD4+FOXP3+T cells has been associated,with either a poor or a good prognosis, or no correlation has beenfound. Here, we demonstrate,in patients with oral squamous cell carcinoma (OSCC), that what really represents a prognostic parameter is not the overall expression of FOXP3 but its intracellular localization.While overallFOXP3 expression in tumor infiltrating CD4+T cells does not correlate with tumor recurrence, its intracellular localization within the CD4 cells does: nuclear FOXP3 (nFOXP3) is associated with tumor recurrence within 3 years, while cytoplasmicFOXP3 (cFOXP3) is associated with a lower likelihood of recurrence. Thus, we propose elevated levels of the cFOXP3/nFOXP3 ratio within tumor infiltrating CD4+ T cells as a predictor of OSCC recurrence. PMID:23977174

  11. Subcellular tracking of drug release from carbon nanotube vehicles in living cells.

    PubMed

    Kang, Bin; Li, Jun; Chang, Shuquan; Dai, Mingzhu; Ren, Chao; Dai, Yaodong; Chen, Da

    2012-03-12

    The direct observation of drug release from carbon nanotube vehicles in living cells is realized through a unique two-dye labeling approach. Single-walled carbon nanotubes (SWNTs) are firstly marked with fluorescein isothiocyanate (FITC) to track their location and movement inside the cell. Then a fluorescent anticancer drug doxorubicin (DOX) is attached by means of π-stacking onto SWNTs. Delivered by SWNTs into cells, DOX will detach from the vehicle in an acidic environment due to the pH-dependent π-π stacking interaction between DOX and SWNTs. From observation of the two different kinds of fluorescence (green and red) that respectively represent the carrier SWNTs and drug DOX, the process of drug release inside the living cell can be monitored under a confocal microscope. Results show that the drug DOX detaches from SWNTs inside the lysosomes to yield free molecules and escape into the cytoplasm and finally into the nucleus, while the vehicle SWNTs are trapped inside the lysosomes, without entering the nucleus. The current observations confirm previously proposed mechanisms for drug/DOX release inside cells. The experimental establishment of drug-release mechanisms in living cells here might provide important insights for future design of new drug-delivery and release systems.

  12. Sperm storage and spermatozoa interaction with epithelial cells in oviduct of Chinese soft-shelled turtle, Pelodiscus sinensis

    PubMed Central

    Chen, Shaofan; Zhang, Linli; Le, Yuan; Waqas, Yasir; Chen, Wei; Zhang, Qian; Ullah, Shakeeb; Liu, Tengfei; Hu, Lisi; Li, Quanfu; Yang, Ping

    2015-01-01

    Spermatozoa are known to be stored within the female genital tract after mating in various species to optimize timing of reproductive events such as copulation, fertilization, and ovulation. The mechanism supporting long-term sperm storage is still unclear in turtles. The aim of this study was to investigate the interaction between the spermatozoa and oviduct in Chinese soft-shelled turtle by light and electron microscopy to reveal the potential cytological mechanism of long-term sperm storage. Spermatozoa were stored in isthmus, uterine, and vagina of the oviduct throughout the year, indicating long-term sperm storage in vivo. Sperm heads were always embedded among the cilia and even intercalated into the apical hollowness of the ciliated cells in the oviduct mucosal epithelium. The stored spermatozoa could also gather in the gland conduit. There was no lysosome distribution around the hollowness of the ciliated cell, suggesting that the ciliated cells of the oviduct can support the spermatozoa instead of phagocytosing them in the oviduct. Immune cells were sparse in the epithelium and lamina propria of oviduct, although few were found inside the blood vessel of mucosa, which may be an indication of immune tolerance during sperm storage in the oviduct of the soft-shelled turtle. These characteristics developed in the turtle benefited spermatozoa survival for a long time as extraneous cells in the oviduct of this species. These findings would help to improve the understanding of reproductive regularity and develop strategies of species conservation in the turtle. The Chinese soft-shelled turtle may be a potential model for uncovering the mechanism behind the sperm storage phenomenon. PMID:26357535

  13. Sperm storage and spermatozoa interaction with epithelial cells in oviduct of Chinese soft-shelled turtle, Pelodiscus sinensis.

    PubMed

    Chen, Shaofan; Zhang, Linli; Le, Yuan; Waqas, Yasir; Chen, Wei; Zhang, Qian; Ullah, Shakeeb; Liu, Tengfei; Hu, Lisi; Li, Quanfu; Yang, Ping

    2015-08-01

    Spermatozoa are known to be stored within the female genital tract after mating in various species to optimize timing of reproductive events such as copulation, fertilization, and ovulation. The mechanism supporting long-term sperm storage is still unclear in turtles. The aim of this study was to investigate the interaction between the spermatozoa and oviduct in Chinese soft-shelled turtle by light and electron microscopy to reveal the potential cytological mechanism of long-term sperm storage. Spermatozoa were stored in isthmus, uterine, and vagina of the oviduct throughout the year, indicating long-term sperm storage in vivo. Sperm heads were always embedded among the cilia and even intercalated into the apical hollowness of the ciliated cells in the oviduct mucosal epithelium. The stored spermatozoa could also gather in the gland conduit. There was no lysosome distribution around the hollowness of the ciliated cell, suggesting that the ciliated cells of the oviduct can support the spermatozoa instead of phagocytosing them in the oviduct. Immune cells were sparse in the epithelium and lamina propria of oviduct, although few were found inside the blood vessel of mucosa, which may be an indication of immune tolerance during sperm storage in the oviduct of the soft-shelled turtle. These characteristics developed in the turtle benefited spermatozoa survival for a long time as extraneous cells in the oviduct of this species. These findings would help to improve the understanding of reproductive regularity and develop strategies of species conservation in the turtle. The Chinese soft-shelled turtle may be a potential model for uncovering the mechanism behind the sperm storage phenomenon.

  14. Red Raspberry Phenols Inhibit Angiogenesis: A Morphological and Subcellular Analysis Upon Human Endothelial Cells.

    PubMed

    Sousa, M; Machado, V; Costa, R; Figueira, M E; Sepodes, B; Barata, P; Ribeiro, L; Soares, R

    2016-07-01

    Polyphenols are a class of natural compounds whose potential as antioxidant, anti-inflammatory, and anti-angiogenesis has been reported in many pathological conditions. Red raspberry extract, rich in polyphenols, has been reported to exert anti-inflammatory effects and prevent cell proliferation in distinct animal models. However, the signaling pathways involved remain unknown. Herein, we used human microvascular endothelial cells (HMVECs) to determine the influence of red raspberry phenolic compound extract concentrations, ranging from 10 to 250 µg gallic acid equivalents (GAE)/mL, on endothelium viability (MTS assay), proliferation (BrdU incorporation), migration (injury assay), and capillary-like structures formation (Matrigel assay). Protein expression in cell lysates was determined by Western blot analysis. We showed that red raspberry extracts reduced cell viability (GI50  = 87,64 ± 6,59 μg GAE/mL) and proliferation in a dose-dependent manner. A significant abrogation of cells ability to migrate to injured areas, even at low concentrations, was observed by injury assay. Cell assembly into capillary-like structures on Matrigel also decreased in a dose dependent-manner for higher extract concentrations, as well as the number of branching points per unit of area. Protein expression analysis showed a dose-dependent decrease in Phospho-VEGFR2 expression, implying abrogation of VEGF signaling activity. We also showed for the first time that red raspberry phenolic compounds induce the rearrangement of filamentous actin cytoskeleton, with an isotropy increase found for higher testing concentrations. Taken together, our findings corroborate the anti-angiogenic potential of red raspberry phenolic compounds and provide new insights into their mode of action upon endothelium. J. Cell. Biochem. 117: 1604-1612, 2016. © 2015 Wiley Periodicals, Inc.

  15. Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells.

    PubMed

    VanLinden, Magali R; Dölle, Christian; Pettersen, Ina K N; Kulikova, Veronika A; Niere, Marc; Agrimi, Gennaro; Dyrstad, Sissel E; Palmieri, Ferdinando; Nikiforov, Andrey A; Tronstad, Karl Johan; Ziegler, Mathias

    2015-11-13

    The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD(+) biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD(+) in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD(+) content, we have expressed plant and yeast mitochondrial NAD(+) carriers in human cells and observed a profound increase in mitochondrial NAD(+). None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD(+) content. Surprisingly, constitutive redistribution of NAD(+) from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD(+) transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD(+) levels. These results suggest that a mitochondrial NAD(+) transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD(+) synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells.

  16. Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles.

    PubMed

    Théry, C; Boussac, M; Véron, P; Ricciardi-Castagnoli, P; Raposo, G; Garin, J; Amigorena, S

    2001-06-15

    Dendritic cells constitutively secrete a population of small (50-90 nm diameter) Ag-presenting vesicles called exosomes. When sensitized with tumor antigenic peptides, dendritic cells produce exosomes, which stimulate anti-tumor immune responses and the rejection of established tumors in mice. Using a systematic proteomic approach, we establish the first extensive protein map of a particular exosome population; 21 new exosomal proteins were thus identified. Most proteins present in exosomes are related to endocytic compartments. New exosomal residents include cytosolic proteins most likely involved in exosome biogenesis and function, mainly cytoskeleton-related (cofilin, profilin I, and elongation factor 1alpha) and intracellular membrane transport and signaling factors (such as several annexins, rab 7 and 11, rap1B, and syntenin). Importantly, we also identified a novel category of exosomal proteins related to apoptosis: thioredoxin peroxidase II, Alix, 14-3-3, and galectin-3. These findings led us to analyze possible structural relationships between exosomes and microvesicles released by apoptotic cells. We show that although they both represent secreted populations of membrane vesicles relevant to immune responses, exosomes and apoptotic vesicles are biochemically and morphologically distinct. Therefore, in addition to cytokines, dendritic cells produce a specific population of membrane vesicles, exosomes, with unique molecular composition and strong immunostimulating properties.

  17. Stem cell differentiation indicated by noninvasive photonic characterization and fractal analysis of subcellular architecture.

    PubMed

    Chalut, Kevin J; Kulangara, Karina; Wax, Adam; Leong, Kam W

    2011-08-01

    We hypothesised that global structural changes in stem cells would manifest with differentiation, and that these changes would be observable with light scattering microscopy. Analysed with a fractal dimension formalism, we observed significant structural changes in differentiating human mesenchymal stem cells within one day after induction, earlier than could be detected by gene expression profiling. Moreover, light scattering microscopy is entirely non-perturbative, so the same sample could be monitored throughout the differentiation process. We explored one possible mechanism, chromatin remodelling, to account for the changes we observed. Correlating with the staining of HP1α, a heterochromatin protein, we applied novel microscopy methods and fractal analysis to monitor the plastic dynamics of chromatin within stem cell nuclei. We showed that the level of chromatin condensation changed during differentiation, and provide one possible explanation for the changes seen with the light scattering method. These results lend physical insight into stem cell differentiation while providing physics-based methods for non-invasive detection of the differentiation process.

  18. How nematode sperm crawl.

    PubMed

    Bottino, Dean; Mogilner, Alexander; Roberts, Tom; Stewart, Murray; Oster, George

    2002-01-15

    Sperm of the nematode, Ascaris suum, crawl using lamellipodial protrusion, adhesion and retraction, a process analogous to the amoeboid motility of other eukaryotic cells. However, rather than employing an actin cytoskeleton to generate locomotion, nematode sperm use the major sperm protein (MSP). Moreover, nematode sperm lack detectable molecular motors or the battery of actin-binding proteins that characterize actin-based motility. The Ascaris system provides a simple 'stripped down' version of a crawling cell in which to examine the basic mechanism of cell locomotion independently of other cellular functions that involve the cytoskeleton. Here we present a mechanochemical analysis of crawling in Ascaris sperm. We construct a finite element model wherein (a) localized filament polymerization and bundling generate the force for lamellipodial extension and (b) energy stored in the gel formed from the filament bundles at the leading edge is subsequently used to produce the contraction that pulls the rear of the cell forward. The model reproduces the major features of crawling sperm and provides a framework in which amoeboid cell motility can be analyzed. Although the model refers primarily to the locomotion of nematode sperm, it has important implications for the mechanics of actin-based cell motility.

  19. Elucidation of the involvement of p14, a sperm protein during maturation, capacitation and acrosome reaction of caprine spermatozoa.

    PubMed

    Nandi, Pinki; Ghosh, Swatilekha; Jana, Kuladip; Sen, Parimal C

    2012-01-01

    Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event.

  20. Conformation of the Bax C-terminus regulates subcellular location and cell death.

    PubMed Central

    Nechushtan, A; Smith, C L; Hsu, Y T; Youle, R J

    1999-01-01

    Bax, a pro-apoptotic member of the Bcl-2 family, translocates from the cytosol to the mitochondria during programmed cell death. We report here that both gain-of-function and loss-of-function mutations can be achieved by altering a single amino acid in the Bax hydrophobic C-terminus. The properly mutated C-terminus of Bax can target a non-relevant protein to the mitochondria, showing that specific conformations of this domain alone allow mitochondrial docking. These data along with N-terminus epitope exposure experiments suggest that the C- and the N-termini interact and that upon triggering of apoptosis, Bax changes conformation, exposing these two domains to insert into the mitochondria and regulate the cell death machinery. PMID:10228148

  1. Efficient production of intersubspecific hybrid mice and embryonic stem cells by intracytoplasmic sperm injection.

    PubMed

    Shinmen, Akie; Honda, Arata; Ohkawa, Mika; Hirose, Michiko; Ogonuki, Narumi; Yuzuriha, Misako; Miki, Hiromi; Mochida, Keiji; Inoue, Kimiko; Abe, Kuniya; Ito, Masao; Ogura, Atsuo

    2007-09-01

    Recently, mice and embryonic stem (ES) cells with allelic polymorphisms have been used extensively in the field of genetics and developmental biology. In this study, we examined whether intersubspecific hybrid mice and ES cells with these genotypes can be efficiently produced by intracytoplasmic sperm injection (ICSI). Frozen-thawed spermatozoa from wild-derived strains, JF1 (Mus musculus molossinus), MSM (M. m. molossinus), HMI (M. m. castaneus), and SWN (M. m. spp.), were directly injected into mature oocytes from laboratory mice ([C57BL/6 x DBA2]F1; M. m. domesticus). The in vitro and in vivo developmental capacity of F1 embryos was not significantly different among the groups (P > 0.05), and term offspring were efficiently obtained in all groups (27%-34% of transferred embryos). However, the mean body and placental weights of the offspring differed significantly with genotype (P < 5 x 10(-10)), with the HMI hybrid greatest in both body and placental weights. In an application study using these F1 offspring, we analyzed their mitochondrial DNA using intersubspecific polymorphisms and found the consistent disappearance of sperm mitochondrial DNA in the F1 progeny. In a second series of experiments, we generated F1 blastocysts by injecting MSM spermatozoa into C57BL/6 oocytes and used them to generate hybrid ES cell lines. The ES cell lines were established at a high efficiency (9 lines from 20 blastocysts) and their allelic polymorphisms were confirmed. Thus, ICSI using cryopreserved spermatozoa allows the efficient and immediate production of a number of F1 hybrid mice and ES cell lines, which can be used for polymorphic analysis of mouse genetics.

  2. Subcellular distribution of ( sup 3 H)-dexamethasone mesylate binding sites in Leydig cells using electron microscope radioautography

    SciTech Connect

    Stalker, A.; Hermo, L.; Antakly, T. )

    1991-01-01

    The present view is that glucocorticoid hormones bind to their cytoplasmic receptors before reaching their nuclear target sites, which include specific DNA sequences. Although it is believed that cytoplasmic sequestration of steroid receptors and other transcription factors (such as NFKB) may regulate the overall activity of these factors, there is little information on the exact subcellular sites of steroid receptors or even of any other transcription factors. Tritiated (3H)-dexamethasone 21-mesylate (DM) is an affinity label that binds covalently to the glucocorticoid receptor (GR), thereby allowing morphological localization of the receptor at the light and electron microscope levels as well as for quantitative radioautographic (RAG) analysis. After injection of 3H-DM into the testis, a specific radioautographic signal was observed in Leydig cells, which correlated with a high level of immunocytochemically demonstrable GR in these cells at the light-microscope level. To localize the 3H-DM binding sites at the electron microscope (EM) level, the testes of 5 experimental and 3 control adrenalectomized rats were injected directly with 20 microCi 3H-DM; control rats received simultaneously a 25-fold excess of unlabeled dexamethasone; 15 min later, rats were fixed with glutaraldehyde and the tissue was processed for EM RAG analysis combined with quantitative morphometry. The radioautographs showed that the cytosol, nucleus, smooth endoplasmic reticulum (sER), and mitochondria were labeled. Since the cytosol was always adjacent to tubules of the sER, the term sER-rich cytosol was used to represent label over sER networks, which may also represent cytosol labeling due to the limited resolution of the radioautographic technique. Labeling was highest in sER-rich cytosol and mitochondria, at 53% and 31% of the total, respectively.

  3. Subcellular calcium measurements in mammalian cells using jellyfish photoprotein aequorin-based probes.

    PubMed

    Bonora, Massimo; Giorgi, Carlotta; Bononi, Angela; Marchi, Saverio; Patergnani, Simone; Rimessi, Alessandro; Rizzuto, Rosario; Pinton, Paolo

    2013-11-01

    The jellyfish Aequorea victoria produces a 22-kDa protein named aequorin that has had an important role in the study of calcium (Ca(2+)) signaling. Aequorin reacts with Ca(2+) via oxidation of the prosthetic group, coelenterazine, which results in emission of light. This signal can be detected by using a special luminescence reader (called aequorinometer) or luminescence plate readers. Here we describe the main characteristics of aequorin as a Ca(2+) probe and how to measure Ca(2+) in different intracellular compartments of animal cells (cytosol, different mitochondrial districts, nucleus, endoplasmic reticulum (ER), Golgi apparatus, peroxisomes and subplasma-membrane cytosol), ranging from single-well analyses to high-throughput screening by transfecting animal cells using DNA vectors carrying recombinant aequorin chimeras. The use of aequorin mutants and modified versions of coelenterazione increases the range of calcium concentrations that can be recorded. Cell culture and transfection takes ∼3 d. An experiment including signal calibration and the subsequent analyses will take ∼1 d.

  4. Lectin binding patterns and carbohydrate mediation of sperm binding to llama oviductal cells in vitro.

    PubMed

    Apichela, Silvana A; Valz-Gianinet, Jorge N; Schuster, Stefanie; Jiménez-Díaz, María A; Roldán-Olarte, Eugenia M; Miceli, Dora C

    2010-04-01

    Sperm binding to oviductal epithelium would be involved in sperm reservoir formation in the utero tubal junction (UTJ). Although in other mammals sperm-oviduct interaction has been proved to be mediated by carbohydrate-recognition mechanisms, the factors implicated in the sperm adhesion to oviductal epithelium of llama are still unknown. In order to assess the role of carbohydrates present in the mucosa surface, we examined the distribution of glycoconjugates in the llama oviduct by confocal lectin-histochemistry. Mannosyl, glucosyl, N-acetylglucosaminyl, galactosyl, N-acetylgalactosaminyl and sialic acid residues were detected in the oviductal mucose glycocalyx. By incubation of UTJ oviductal explants with LCA, DBA, UEA-1 or PNA lectin previous to co-culture with sperm, we observed a significant decrease in sperm binding only with LCA lectin. In the mucosa surface there were numerous d-glucosyl and D-manosyl residues, which were spotted by this lectin. Probably, this fact promotes the whole covering of the oviduct luminal surface by the sugar-lectin complex, preventing sperm access and adhesion of further residues. However, sperm incubation with mannose or glucose does not significantly prevent binding, which means that glucose and mannose would not be involved in a specific sperm-oviduct interaction. On the other hand, we observed a high reduction in sperm binding to UTJ explants with N-acetylgalactosamine and galactose (p<0.001). Coincidentally, binding sites for N-acetylgalactosamine-PAA-FITC conjugate were observed on the whole surface of the sperm, supporting the concept that llama sperm have lectin-like molecules in their surface, as is the case in other mammals. Probably, these lectin-like molecules, by means of N-acetylgalactosamine and galactose recognition, could link the sperm to the oviductal mucosa with the purpose of forming storing sites in the UTJ. Our results support the idea that more than one carbohydrate could participate in sperm reservoir

  5. Detection and bioimaging of nitric oxide in bovine oocytes and sperm cells.

    PubMed

    Reyes, R; Vázquez, M L S; Delgado, N M

    2004-01-01

    Mammalian gametes contain constitutive nitric oxide synthases (NOS) to synthesize nitric oxide (NO). The detection and bioimaging of NO in bovine gametes is important to determine the regulatory roles of NO during the different events of fertilization. Diaminofluoresceins, new fluorescence indicators for NO, were applied to detect the release of NO from bovine gametes. These compounds yield green fluorescent triazolofluoresceins, which provide the advantages of high specificity, sensitivity, and simplicity for the detection of NO. In this study, we mapped the expression of NOS in the bovine sperm and ova. NOS activity in sperm first appeared in the acrosome, then 60 min later in the head, middle piece, cytoplasmic droplet, and tail. Cow ova had high NO activity in the cytoplasm and in the surrounding corona cells, but not in the zona pellucida. These results show that for bovine gametes, the synthesis NO by the NOS system presents clear patterns of time and spatial distribution that may be important for the different events of fertilization.

  6. Sperm proteome and reproductive technologies in mammals.

    PubMed

    Li, Chun-Jin; Wang, Dong; Zhou, Xu

    2016-10-01

    Sperm is highly differentiated cell that can be easily obtained and purified. Mature sperm is considered to be transcriptionally and translationally silent and incapable of protein synthesis. Recently, a large number of proteins have been identified in sperm from different species by using the proteomic approaches. Clinically, sperm proteins can be used as markers for male infertility due to different protein profiles identified in sperm from fertile and infertile male animals. Recent evidences have shown that the conditions of sperm preservation in vitro can also change the sperm protein profiles. This paper reviews the recent scientific publications available to address sperm proteome and their relationship with sperm cryopreservation, capacitation, fertilization, and separation of X and Y sperm. Future directions in the application of sperm proteomics to develop or optimize reproductive technologies in mammals are also discussed.

  7. Increased expression with differential subcellular location of cytidine deaminase APOBEC3G in human CD4(+) T-cell activation and dendritic cell maturation.

    PubMed

    Oliva, Harold; Pacheco, Rodrigo; Martinez-Navio, José M; Rodríguez-García, Marta; Naranjo-Gómez, Mar; Climent, Núria; Prado, Carolina; Gil, Cristina; Plana, Montserrat; García, Felipe; Miró, José M; Franco, Rafael; Borras, Francesc E; Navaratnam, Naveenan; Gatell, José M; Gallart, Teresa

    2016-08-01

    APOBEC3G (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G; A3G) is an innate defense protein showing activity against retroviruses and retrotransposons. Activated CD4(+) T cells are highly permissive for HIV-1 replication, whereas resting CD4(+) T cells are refractory. Dendritic cells (DCs), especially mature DCs, are also refractory. We investigated whether these differences could be related to a differential A3G expression and/or subcellular distribution. We found that A3G mRNA and protein expression is very low in resting CD4(+) T cells and immature DCs, but increases strongly following T-cell activation and DC maturation. The Apo-7 anti-A3G monoclonal antibody (mAb), which was specifically developed, confirmed these differences at the protein level and disclosed that A3G is mainly cytoplasmic in resting CD4(+) T cells and immature DCs. Nevertheless, A3G translocates to the nucleus in activated-proliferating CD4(+) T cells, yet remaining cytoplasmic in matured DCs, a finding confirmed by immunoblotting analysis of cytoplasmic and nuclear fractions. Apo-7 mAb was able to immunoprecipitate endogenous A3G allowing to detect complexes with numerous proteins in activated-proliferating but not in resting CD4(+) T cells. The results show for the first time the nuclear translocation of A3G in activated-proliferating CD4(+) T cells.

  8. Expression of a P-selectin ligand in zona pellucida of porcine oocytes and P-selectin on acrosomal membrane of porcine sperm cells. Potential implications for their involvement in sperm-egg interactions.

    PubMed

    Geng, J G; Raub, T J; Baker, C A; Sawada, G A; Ma, L; Elhammer, A P

    1997-05-05

    The selectin family of cell adhesion molecules mediates initial leukocyte adhesion to vascular endothelial cells at sites of inflammation. O-glycan structural similarities between oligosaccharides from human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and from zona pellucida glycoproteins of porcine oocytes indicate the possible existence of a P-selectin ligand in the zona pellucida. Here, using biochemical as well as morphological approaches, we demonstrate that a P-selectin ligand is expressed in the porcine zona pellucida. In addition, a search for a specific receptor for this ligand leads to the identification of P-selectin on the acrosomal membrane of porcine sperm cells. In vitro binding of porcine acrosome-reacted sperm cells to oocytes was found to be Ca2+ dependent and inhibitable with either P-selectin, P-selectin receptor-globulin, or leukocyte adhesion blocking antibodies against P-selectin and PSGL-1. Moreover, porcine sperm cells were found to be capable of binding to human promyeloid cell line HL-60. Taken together, our findings implicate a potential role for the oocyte P-selectin ligand and the sperm P-selectin in porcine sperm-egg interactions.

  9. Characterization and sub-cellular localization of GalNAc-binding proteins isolated from human hepatic stellate cells.

    PubMed

    Zhong, Yaogang; Zhang, Jing; Yu, Hanjie; Zhang, Jiaxu; Sun, Xiu-Xuan; Chen, Wentian; Bian, Huijie; Li, Zheng

    2015-12-25

    Although the expression levels of total GalNAc-binding proteins (GNBPs) were up-regulated significantly in human hepatic stellate cells (HSCs) activated with transforming growth factor-β1(TGF-β1), yet little is known about the precise types, distribution and sub-cellular localization of the GNBPs in HSCs. Here, 264 GNBPs from the activated HSCs and 257 GNBPs from the quiescent HSCs were identified and annotated. A total of 46 GNBPs were estimated to be significantly up-regulated and 40 GNBPs were estimated to be significantly down-regulated in the activated HSCs. For example, the GNBPs (i.e. BTF3, COX17, and ATP5A1) responsible for the regulation of protein binding were up-regulated, and those (i.e. FAM114A1, ENO3, and TKT) responsible for the regulation of protein binding were down-regulated in the activated HSCs. The motifs of the isolated GNBPs showed that Proline residue had the maximum preference in consensus sequences. The western blotting showed the expression levels of COX17, and PRMT1 were significantly up-regulated, while, the expression level of CLIC1(B5) was down-regulated in the activated HSCs and liver cirrhosis tissues. Moreover, the GNBPs were sub-localized in the Golgi apparatus of HSCs. In conclusion, the precision alteration of the GNBPs referred to pathological changes in liver fibrosis/cirrhosis may provide useful information to find new molecular mechanism of HSC activation and discover the biomarkers for diagnosis of liver fibrosis/cirrhosis as well as development of new anti-fibrotic strategies.

  10. Efficient Subcellular Targeting to the Cell Nucleus of Quantum Dots Densely Decorated with a Nuclear Localization Sequence Peptide.

    PubMed

    Maity, Amit Ranjan; Stepensky, David

    2016-01-27

    development of subcellularly targeted DDSs that will deliver specific drugs to the nuclei of the target cells and will enhance efficacy and reduce toxicity of these drugs.

  11. Subcellular localization of RNAs in transfected cells: role of sequences at the 5' terminus.

    PubMed Central

    Alfonso-Pizarro, A; Carlson, D P; Ross, J

    1984-01-01

    We have investigated the intracellular location of RNAs transcribed from transfected DNA. COS cells transfected with a clone containing the human adult beta globin gene contain three classes of globin RNAs. Their 3' termini and splice sites are indistinguishable from those of mature reticulocyte beta globin mRNA, and they are polyadenylated. However, as determined by S1 mapping, their 5' sequences are different. The 5' terminus of one is the same as that of mature beta globin mRNA (+1, cap site). The presumed 5' terminus of the second is located 30 nucleotides downstream from the cap site (+30). The third class contains additional nucleotides transcribed from sequences located 5' to the cap site (5' upstream RNA). The 5' upstream RNA molecules are restricted to the nucleus and are more stable than heterogeneous nuclear RNA. The +30 and +1 RNAs are located primarily in the cytoplasm. The data support the notion that nucleotide sequences and/or secondary modifications in the 5' region determine if an RNA is to be transported. Images PMID:6209612

  12. Sperm-cell ultrastructure of North American sturgeons. IV. The pallid sturgeon (Scaphirhynchus albus Forbes and Richardson, 1905)

    USGS Publications Warehouse

    DiLauro, M.N.; Walsh, R.A.; Peiffer, M.; Bennett, R.M.

    2001-01-01

    Sperm-cell morphology and ultrastructure in the pallid sturgeon (Scaphirhynchus albus) were examined using transmission and scanning electron microscopy. Metrics and structure were compared with similar metrics obtained from other published descriptions of sturgeon sperm cells. General morphology was found to be similar to that of sperm cells of the white (Acipenser transmontanus), lake (A. fulvescens), stellate (A. stellatus), Chinese (A. sinensis), Russian (A. gueldenstaedti colchicus), and shortnose (A. brevirostrum) sturgeons, which all shared a gradual tapering of the nuclear diameter from posterior to anterior, unlike that of the Atlantic sturgeon (A. oxyrhynchus). The sperm cell of the pallid sturgeon was similar in size to that of the Atlantic sturgeon, being only slightly larger. The sperm cell of the pallid sturgeon differed from those of other sturgeons chiefly in the acrosomal region, where the posterolateral projections (PLP) have the shape of an acute triangle and are arranged in a spiral about the longitudinal axis of the cell. The PLP were longer than those of other sturgeons, being twice the length of those of the Atlantic sturgeon and 58% longer than those of the lake sturgeon. Also, in cross section the acrosome had the shape of a hollow cone rather than the cap of an oak tree acorn, as was found in ultrastructural studies of other sturgeons. In addition, we were able to confirm that the structural arrangement of the distal centriole of the midpiece is identical with that of the proximal centriole: nine sets of microtubular triplets around the periphery of the centriole. This information is of potential use to fishery biologists, forensic biologists, zoologists, reproductive physiologists, taxonomists, evolutionary biologists, and aquaculturists.

  13. Tropomyosin variants describe distinct functional subcellular domains in differentiated vascular smooth muscle cells.

    PubMed

    Gallant, Cynthia; Appel, Sarah; Graceffa, Philip; Leavis, Paul; Lin, Jim Jung-Ching; Gunning, Peter W; Schevzov, Galina; Chaponnier, Christine; DeGnore, Jon; Lehman, William; Morgan, Kathleen G

    2011-06-01

    Tropomyosin (Tm) is known to be an important gatekeeper of actin function. Tm isoforms are encoded by four genes, and each gene produces several variants by alternative splicing, which have been proposed to play roles in motility, proliferation, and apoptosis. Smooth muscle studies have focused on gizzard smooth muscle, where a heterodimer of Tm from the α-gene (Tmsm-α) and from the β-gene (Tmsm-β) is associated with contractile filaments. In this study we examined Tm in differentiated mammalian vascular smooth muscle (dVSM). Liquid chromatography-tandem mass spectrometry (LC MS/MS) analysis and Western blot screening with variant-specific antibodies revealed that at least five different Tm proteins are expressed in this tissue: Tm6 (Tmsm-α) and Tm2 from the α-gene, Tm1 (Tmsm-β) from the β-gene, Tm5NM1 from the γ-gene, and Tm4 from the δ-gene. Tm6 is by far most abundant in dVSM followed by Tm1, Tm2, Tm5NM1, and Tm4. Coimmunoprecipitation and coimmunofluorescence studies demonstrate that Tm1 and Tm6 coassociate with different actin isoforms and display different intracellular localizations. Using an antibody specific for cytoplasmic γ-actin, we report here the presence of a γ-actin cortical cytoskeleton in dVSM cells. Tm1 colocalizes with cortical cytoplasmic γ-actin and coprecipitates with γ-actin. Tm6, on the other hand, is located on contractile bundles. These data indicate that Tm1 and Tm6 do not form a classical heterodimer in dVSM but rather describe different functional cellular compartments.

  14. Why so many sperm cells? Not only a possible means of mitigating the hazards inherent to human reproduction but also an indicator of an exaptation

    PubMed Central

    Barlow, Peter W.

    2016-01-01

    ABSTRACT Redundancy—the excess of supply over necessity—has recently been proposed for human sperm cells. However, the apparent superfluity of cell numbers may be necessary in order to circumvent the hazards, many of which can be quantified, that can occur during the transition from gametogenesis within the testes to zygosis within the female reproductive tract. Sperm cell numbers are directly related to testicular volume, and it is owing to a redundancy, and the possible exaptation, of this latter parameter that a putative excess of sperm cells is perceived. PMID:27574542

  15. Ca exchange under non-perfusion-limited conditions in rat ventricular cells: Identification of subcellular compartments

    SciTech Connect

    Langer, G.A.; Rich, T.L.; Orner, F.B. )

    1990-08-01

    Freshly prepared ventricular myocytes from rat hearts, aliquots of which were tested for sarcolemmal integrity by La exposure, were labeled at high 45Ca specific activity. Isotope was subsequently washed out at a perfusion rate of 2.8 ml/s with washout solution sampled each 1 s. No initial unrecorded period of washout was imposed. Four compartments were distinguishable: (1) a rapid compartment (RC) containing 2.6 mmol Ca/kg dry wt of La-displaceable Ca, half time (t1/2) less than 1 s; (2) an intermediate compartment(s) (IC) containing 2.1 mmol, t1/2 = 3 and 19 s; (3) a slow compartment (SC) containing 1.6 mmol, t1/2 = 3.6 min; (4) an inexchangeable compartment that demonstrated no 45Ca uptake after 60-min labeling containing 1.2 mmol. Introduction of 10 mM caffeine as a probe for sarcoplasmic reticulum (SR) content at various times during the washouts caused an increased release of 45Ca. The net increased 45Ca release plotted as a function of time at which caffeine was introduced produced a biexponential curve with t1/2s of 2 and 22 s, very similar to the t1/2s of the IC. Ryanodine (1 microM) significantly reduced the caffeine-induced 45Ca release, confirming the SR locus of the IC. Cells were perfused with 10 mM NaH2PO4 to specifically increase mitochondrial 45Ca labeling. Subsequent removal of PO4 at various times during washouts produced large increases in effluent 45Ca. A plot of the net peak release of 45Ca vs. time of PO4 removal was monoexponential with t1/2 = 3.3 min, very similar to the SC t1/2. The large La-accessible RC remains unlocalized, but the rapidity of its exchange places it in the sarcolemma and/or at sites in rapid equilibrium with the sarcolemma.

  16. Sperm Patch-Clamp

    PubMed Central

    Lishko, Polina; Clapham, David E.; Navarro, Betsy; Kirichok, Yuriy

    2014-01-01

    Sperm intracellular pH and calcium concentration ([Ca2+]i) are two central factors that control sperm activity within the female reproductive tract. As such, the ion channels of the sperm plasma membrane that alter intracellular sperm [Ca2+] and pH play important roles in sperm physiology and the process of fertilization. Indeed, sperm ion channels regulate sperm motility, control sperm chemotaxis toward the egg in some species, and may trigger the acrosome reaction. Until recently, our understanding of these important molecules was rudimentary due to the inability to patch-clamp spermatozoa and directly record the activity of these ion channels under voltage clamp. Recently, we overcame this technical barrier and developed a method for reproducible application of the patch-clamp technique to mouse and human spermatozoa. This chapter covers important aspects of application of the patch-clamp technique to spermatozoa, such as selection of the electrophysiological equipment, isolation of spermatozoa for patch-clamp experiments, formation of the gigaohm seal with spermatozoa, and transition into the whole-cell mode of recording. We also discuss potential pitfalls in application of the patch-clamp technique to flagellar ion channels. PMID:23522465

  17. Production of fertile sperm from in vitro propagating enriched spermatogonial stem cells of farmed catfish, Clarias batrachus.

    PubMed

    Nayak, Swapnarani; Ferosekhan, Shajahan; Sahoo, Sangram Ketan; Sundaray, Jitendra Kumar; Jayasankar, Pallipuram; Barman, Hirak Kumar

    2016-12-01

    Spermatogenesis is a highly co-ordinated and complex process. In vitro propagation of spermatogonial stem cells (SSCs) could provide an avenue in which to undertake in vivo studies of spermatogenesis. Very little information is known about the SSC biology of teleosts. In this study, collagenase-treated testicular cells of farmed catfish (Clarias batrachus, popularly known as magur) were purified by Ficoll gradient centrifugation followed by magnetic activated cell sorting using Thy1.2 (CD90.2) antibody to enrich for the spermatogonial cell population. The sorted spermatogonial cells were counted and gave ~3 × 106 cells from 6 × 106 pre-sorted cells. The purified cells were cultured in vitro for >2 months in L-15 medium containing fetal bovine serum (10%), carp serum (1%) and other supplements. Microscopic observations depicted typical morphological SSC features, bearing a larger nuclear compartment (with visible perinuclear bodies) within a thin rim of cytoplasm. Cells proliferated in vitro forming clumps/colonies. mRNA expression profiling by qPCR documented that proliferating cells were Plzf + and Pou2+, indicative of stem cells. From 60 days onwards of cultivation, the self-renewing population differentiated to produce spermatids (~6 × 107 on day 75). In vitro-produced sperm (2260 sperm/SSC) were free swimming in medium and hence motile (non-progressive) in nature. Of those, 2% were capable of fertilizing and generated healthy diploid fingerlings. Our documented evidence provides the basis for producing fertile magur sperm in vitro from cultured magur SSCs. Our established techniques of SSC propagation and in vitro sperm production together should trigger future in vivo experiments towards basic and applied biology research.

  18. Disrupting the wall accumulation of human sperm cells by artificial corrugation

    PubMed Central

    Jeyaram, Y.; Condat, C. A.; Oviedo, M.; Berdakin, I.; Moshchalkov, V. V.; Giojalas, L. C.; Silhanek, A. V.; Marconi, V. I.

    2015-01-01

    Many self-propelled microorganisms are attracted to surfaces. This makes their dynamics in restricted geometries very different from that observed in the bulk. Swimming along walls is beneficial for directing and sorting cells, but may be detrimental if homogeneous populations are desired, such as in counting microchambers. In this work, we characterize the motion of human sperm cells ∼60 μm long, strongly confined to ∼25 μm shallow chambers. We investigate the nature of the cell trajectories between the confining surfaces and their accumulation near the borders. Observed cell trajectories are composed of a succession of quasi-circular and quasi-linear segments. This suggests that the cells follow a path of intermittent trappings near the top and bottom surfaces separated by stretches of quasi-free motion in between the two surfaces, as confirmed by depth resolved confocal microscopy studies. We show that the introduction of artificial petal-shaped corrugation in the lateral boundaries removes the tendency of cells to accumulate near the borders, an effect which we hypothesize may be valuable for microfluidic applications in biomedicine. PMID:26015834

  19. Sperm fucosyltransferase-5 mediates spermatozoa-oviductal epithelial cell interaction to protect human spermatozoa from oxidative damage.

    PubMed

    Huang, Venus Wenxin; Lee, Cheuk-Lun; Lee, Yin-Lau; Lam, Kevin K W; Ko, Jennifer K Y; Yeung, William S B; Ho, Pak-Chung; Chiu, Philip C N

    2015-06-01

    Oxidative damage by reactive oxygen species (ROS) is a major cause of sperm dysfunction. Excessive ROS generation reduces fertilization and enhances DNA damage of spermatozoa. Interaction between spermatozoa and oviductal epithelial cells improves the fertilizing ability of and reduces chromatin damage in spermatozoa. Our previous data showed that oviductal epithelial cell membrane proteins interact with the human spermatozoa and protect them from ROS-induced reduction in sperm motility, membrane integrity and DNA integrity. Sperm fucosyltransferase-5 (sFUT5) is a membrane carbohydrate-binding protein on human spermatozoa. In this study, we demonstrate for the first time that sFUT5 is involved in human spermatozoa-oviduct interaction and the beneficial effects of such interaction on the fertilizing ability of human spermatozoa. Anti-sFUT5 antibody-treated spermatozoa had reduced binding to oviductal membrane proteins. It is consistent with the result that affinity-purified sFUT5 is bound to the epithelial lining of human oviduct and to the immortalized human oviductal epithelial cell line, OE-E6/E7. Pretreatment of spermatozoa with anti-sFUT5 antibody and oviductal membrane proteins with sFUT5 suppressed the protective action of oviductal membrane proteins against ROS/cryopreservation-induced oxidative damage in spermatozoa. Asialofetuin, a reported sFUT5 substrate, can partly mimic the protective effect of oviductal epithelial cell membrane proteins on sperm motility, membrane and DNA integrity. The results enhance our understanding on the protective mechanism of oviduct on sperm functions.

  20. Zinc-silicon interactions influencing sperm chromatin integrity and testicular cell development in the rat as measured by flow cytometry.

    PubMed

    Evenson, D P; Emerick, R J; Jost, L K; Kayongo-Male, H; Stewart, S R

    1993-04-01

    Flow-cytometric procedures were used to determine effects of dietary Zn and Si variations on rat testicular cell development, including integrity of caudal epididymal sperm chromatin structure defined as the susceptibility of DNA to denaturation in situ. Concentrations of 4 (deficient), 12 (adequate), and 500 (excessive) mg of Zn/kg of diet were used with Si concentrations of 0 (low), 540 (medium), and 2,700 (high) mg/kg of diet in a 3 x 3 factorial arrangement. Three-week-old Sprague-Dawley male rats were fed the experimental diets for 8 wk. Rats fed the Zn-deficient/Si-low diet demonstrated significant deviations in the ratio of testicular cell types present, including a reduction of S phase and total haploid cells. Furthermore, approximately 50% of epididymal sperm had a significant decrease in resistance to DNA denaturation in situ. In the Zn-deficient/Si-medium treatment, the effects of Si on animal and testicular growth, distribution of testicular cell types, and sperm chromatin structure integrity were quite similar to the effects of the Zn-adequate diets. A toxic effect of Zn on sperm chromatin structure integrity observed in the Zn-excess/Si-medium treatment seemed to be counteracted by Si in the Zn-excess/Si-high treatment. Silicon at medium and high levels seems to affect Zn metabolism through potentiation and antagonistic reactions, respectively. Zinc deficiency likely disrupts the normal sperm chromatin quaternary structure in which Zn plays a role by providing stability and resistance to DNA denaturation in situ.

  1. The irradiation of rabbit sperm cells with He-Ne laser prevents their in vitro liquid storage dependent damage.

    PubMed

    Iaffaldano, Nicolaia; Rosato, Maria Pina; Paventi, Gianluca; Pizzuto, Roberto; Gambacorta, Mario; Manchisi, Angelo; Passarella, Salvatore

    2010-05-01

    The aim of the study was to investigate the effects of different energy doses of helium-neon (He-Ne) laser irradiation on both mitochondrial bioenergetics functions and functional quality of rabbit spermatozoa during 48 h of in vitro liquid storage at 15 degrees C. 11 rabbit semen pools were each divided into four aliquots: three of them were irradiated with He-Ne laser with different energy doses (3.96, 6.12 and 9.00 J/cm(2)) being the last control kept under the same experimental conditions without irradiation. Sperm motility, viability and acrosome integrity were monitored together with cytochrome c oxidase (COX) activity and the cell energy charge (EC) at 0, 24 and 48 h of storage. Irradiated samples stored for 24 and 48 h better maintained motility (P < 0.01), acrosome integrity (P < 0.01) and viability (P < 0.05) with respect to the control, particularly with the energy dose of 6.12 J/cm(2) that showed the most intense biostimulative effect. COX activity and EC were immediately increased by irradiation particularly in the treatments 6.12 and 9.00 J/cm(2) (P < 0.05), that maintained their levels higher with respect to the control after 48 h of storage (P < 0.01). COX activity of rabbit sperm cells was positively correlated with EC (P < 0.05), viability (P < 0.01) and acrosome integrity (P < 0.05) parameters. These results indicate that the effects of He-Ne laser irradiation on sperm cells are mediated through the stimulation of the sperm mitochondrial respiratory chain and that this effect plays a significant role in the augmentation of the rabbit sperm cells' capability to survive during liquid storage conditions.

  2. Segregation of sperm mitochondria in two- and four-cell embryos of the blue mussel Mytilus edulis: Implications for the mechanism of doubly uniparental inheritance of mitochondrial DNA.

    PubMed

    Cogswell, Andrew T; Kenchington, Ellen L R; Zouros, Eleftherios

    2006-07-01

    Species of the family Mytilidae have 2 mitochondrial genomes, one that is transmitted through the egg and one that is transmitted through the sperm. In the Mytilus edulis species complex (M. edulis, M. galloprovincialis, and M. trossulus) there is also a strong mother-dependent sex-ratio bias in favor of one or the other sex among progeny from pair matings. In a previous study, we have shown that sperm mitochondria enter the egg and that their behavior during cell division is different depending on whether the egg originated from a female- or male-biased mother. Specifically, in eggs from females that produce mostly or exclusively daughters, sperm mitochondria disperse randomly among cells after egg division. In eggs from females that produce predominantly sons, sperm mitochondria tend to stay together in the same cell. Here, we extend these observations and show that in 2- and 4-cell embryos from male-biased mothers most sperm mitochondria are located near or at the cleavage furrow of the major cell, in contrast to embryos from female-biased mothers where there is no preferential association of sperm mitochondria with the cleavage furrow. This observation provides evidence for an early developmental mechanism through which sperm mitochondria are preferentially channeled into the primordial cells of male embryos, thus making the paternal mitochondrial genome the dominant mtDNA component of the male germ line.

  3. Does blood transfusion affect pituitary gonadal axis and sperm parameters in young males with sickle cell disease?

    PubMed Central

    Soliman, Ashraf T.; Yasin, Mohamed; El-Awwa, Ahmed; Abdelrahman, Mohamed O.; De Sanctis, Vincenzo

    2013-01-01

    Objective: We evaluated the effect of packed red cell transfusion (PCTx) on serum concentrations of gonadotropins luteinizing hormone and follicle-stimulating hormone (LH and FSH) and testosterone (T) levels and measured sperm parameters in young adults with sickle cell disease (SCD) on top-up transfusion (TTx) and those on exchange transfusion (ETx) regimen. Materials and Methods: Basal serum concentrations of FSH, LH, and T and semen parameters were evaluated before and 7 days after PCTx in 18 young adults with transfusion-dependent SCD, aged 20.7 ± 2.88 years. They had full pubertal development (Tanner's stage 5), and capacity to ejaculate. They were regularly transfused since early childhood. Chelation therapy was started early during the first 2 years of life using desferrioxamine and was replaced by deferasirox for the last 4-5 years. Ten patients were on TTx and eight were on ETx regimen. Results: PCTx significantly increased hemoglobin (Hb) from 8.5 ± 1.17 g/dl to 10.5 ± 0.4 g/dl, T from 12.3 ± 1.24 nmol/L to 14.23 ± 1.22 nmol/L and gonadotropins’ concentrations. Sperm parameters improved significantly after PCTx including: total sperm count from 87.4 ± 24.6 million/ml to 146.2 ± 51.25 million/ml, total progressive sperm motility (TPM) from 40.8 ± 11.1 million/ml to 93.4 ± 38.3 million/ml, rapid progressive sperm motility (RPM) progressive motility from 29.26 ± 8.75 million/ml to 67.4 ± 29 million/ml. After PCTx the total sperm count, TPM and RPM were significantly better in the ETx group versus the TTx group. Before and after PCTx, T concentrations were correlated significantly with sperm total count, volume, TPM and RPM (r = 0.53, 0.55, 0.42, and 0.38, respectively, P < 0.01). Hb concentrations were correlated significantly with sperm count, TPM, RPM, and % of sperms with normal morphology (r = 0.60, 0.69, 0.66, and 0.86, respectively, P < 0.001). Conclusion: Our study suggests that in males with SCD blood transfusion is associated with

  4. Subzero water permeability parameters and optimal freezing rates for sperm cells of the southern platyfish, Xiphophorus maculatus.

    PubMed

    Pinisetty, D; Huang, C; Dong, Q; Tiersch, T R; Devireddy, R V

    2005-06-01

    This study reports the subzero water transport characteristics (and empirically determined optimal rates for freezing) of sperm cells of live-bearing fishes of the genus Xiphophorus, specifically those of the southern platyfish Xiphophorus maculatus. These fishes are valuable models for biomedical research and are commercially raised as ornamental fish for use in aquariums. Water transport during freezing of X. maculatus sperm cell suspensions was obtained using a shape-independent differential scanning calorimeter technique in the presence of extracellular ice at a cooling rate of 20 degrees C/min in three different media: (1) Hanks' balanced salt solution (HBSS) without cryoprotective agents (CPAs); (2) HBSS with 14% (v/v) glycerol, and (3) HBSS with 10% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder with a length of 52.35 microm and a diameter of 0.66 microm with an osmotically inactive cell volume (Vb) of 0.6 V0, where V0 is the isotonic or initial cell volume. This translates to a surface area, SA to initial water volume, WV ratio of 15.15 microm(-1). By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best fit membrane permeability parameters (reference membrane permeability to water at 0 degrees C, Lpg or Lpg [cpa] and the activation energy, E(Lp) or E(Lp) [cpa]) were found to range from: Lpg or Lpg [cpa] = 0.0053-0.0093 microm/minatm; E(Lp) or E(Lp) [cpa] = 9.79-29.00 kcal/mol. By incorporating these membrane permeability parameters in a recently developed generic optimal cooling rate equation (optimal cooling rate, [Formula: see text] where the units of B(opt) are degrees C/min, E(Lp) or E(Lp) [cpa] are kcal/mol, L(pg) or L(pg) [cpa] are microm/minatm and SA/WV are microm(-1)), we determined the optimal rates of freezing X. maculatus sperm cells to be 28 degrees C/min (in HBSS), 47 degrees C/min (in HBSS+14% glycerol) and 36 degrees C/min (in HBSS+10% DMSO). Preliminary empirical

  5. SLC6 family transporter SNF-10 is required for protease-mediated activation of sperm motility in C. elegans

    PubMed Central

    Fenker, Kristin E.; Hansen, Angela A.; Chong, Conrad A.; Jud, Molly C.; Duffy, Brittany A.; Norton, J. Paul; Hansen, Jody M.; Stanfield, Gillian M.

    2014-01-01

    Summary Motility of sperm is crucial for their directed migration to the egg. The acquisition and modulation of motility are regulated to ensure that sperm move when and where needed, thereby promoting reproductive success. One specific example of this phenomenon occurs during differentiation of the amoeboid sperm of C. elegans as they activate from a round spermatid to a mature, crawling spermatozoon. Sperm activation is regulated by redundant pathways to occur at a specific time and place for each sex. Here, we report the identification of the solute carrier 6 (SLC6) transporter protein SNF-10 as a key regulator of C. elegans sperm activation in response to male protease activation signals. We find that SNF-10 is present in sperm and is required for activation by the male but not by the hermaphrodite. Loss of both snf-10 and a hermaphrodite activation factor render sperm completely insensitive to activation. Using in vitro assays, we find that snf-10 mutant sperm show a specific deficit in response to protease treatment but not to other activators. Prior to activation, SNF-10 is present in the plasma membrane, where it represents a strong candidate to receive signals that lead to subcellular morphogenesis. After activation, it shows polarized localization to the cell body region that is dependent on membrane fusions mediated by the dysferlin FER-1. Our discovery of snf-10 offers insight into the mechanisms differentially employed by the two sexes to accomplish the common goal of producing functional sperm, as well as how the physiology of nematode sperm may be regulated to control motility as it is in mammals. PMID:24929237

  6. Quantitative subcellular imaging of boron compounds in individual mitotic and interphase human glioblastoma cells with imaging secondary ion mass spectrometry (SIMS).

    PubMed

    Chandra, S; Tjarks, W; Lorey, D R; Barth, R F

    2008-01-01

    Boron measurements at subcellular scale are essential in boron neutron capture therapy (BNCT) of cancer as the nuclear localization of boron-10 atoms can enhance the effectiveness of killing individual tumour cells. Since tumours contain a heterogeneous population of cells in interphase as well as in the M phase (mitotic division) of the cell cycle, it is important to evaluate the subcellular distribution of boron in both phases. In this work, the secondary ion mass spectrometry (SIMS) based imaging technique of ion microscopy was used to quantitatively image boron from two BNCT agents, clinically used p-boronophenylalanine (BPA) and 3-[4-(o-carboran-1-yl)butyl]thymidine (N4), in mitotic metaphase and interphase human glioblastoma T98G cells. N4 belongs to a class of experimental BNCT agents, designated 3-carboranyl thymidine analogues (3CTAs), which presumably accumulate selectively in cancer cells due to a process referred to as kinase-mediated trapping (KMT). The cells were exposed to BPA for 1 h and N4 for 2 h. A CAMECA IMS-3f SIMS ion microscope instrument capable of producing isotopic images with 500 nm spatial resolution was used in the study. Observations were made in cryogenically prepared fast frozen, and freeze-fractured, freeze-dried cells. Three discernible subcellular regions were studied: the nucleus, a characteristic mitochondria-rich perinuclear cytoplasmic region, and the remaining cytoplasm in interphase T98G cells. In metaphase cells, the chromosomes and the cytoplasm were studied for boron localization. Intracellular concentrations of potassium and sodium also were measured in each cell in which the subcellular boron concentrations were imaged. Since the healthy cells maintain a K/Na ratio of approximately 10 due to the presence of Na-K-ATPase in the plasma membrane of mammalian cells, these measurements provided validation for cryogenic sample preparation and indicated the analysis healthy, well preserved cells. The BPA-treated interphase

  7. Effect of aracnotoxin from Latrodectus mactans on bovine sperm function: modulatory action of bovine oviduct cells and their secretions.

    PubMed

    Gómez, P N; Alvarez, J G; Parodi, J; Romero, F; Sánchez, R

    2012-05-01

    Latrodectus mactans' aracnotoxin (Atx) induces changes in sperm function that could be used as a co-adjuvant in male contraceptive barrier methods. This effect includes the suppression of intracellular reactive oxygen species (ROS), an event necessary for capacitation, chemotaxis and acrosome reaction (AR). The sperm that are not trapped by the barrier method can reach the oviduct before fertilisation and be exposed to the secretions of the oviducts. This study evaluated the effect of bovine tubal explants (TU) and conditioned media (CM) from the ampullar and isthmal regions on spermatozoa exposed to Atx. Thawed bovine sperm were incubated with Atx, TU and CM from the ampullar and isthmal regions for 4 h and then DNA integrity, intracellular ROS and lysophosphatidylcholine-induced AR were determined. Spermatozoa exposed to Atx and co-incubated with TU and CM for 4 h produced an increase in sperm DNA damage, a decrease in ROS production and a decrease in %AR, compared with the control. A similar result was obtained from the co-incubation of spermatozoa with Atx. In conclusion, the effect of Atx is not modified by tubal cells or their secretions and this opens the door to future studies to evaluate the application of synthetic peptides obtained from Atx as a co-adjuvant of contraceptive barrier methods.

  8. Vegetative and sperm cell-specific aquaporins of Arabidopsis highlight the vacuolar equipment of pollen and contribute to plant reproduction.

    PubMed

    Wudick, Michael M; Luu, Doan-Trung; Tournaire-Roux, Colette; Sakamoto, Wataru; Maurel, Christophe

    2014-04-01

    The water and nutrient status of pollen is crucial to plant reproduction. Pollen grains of Arabidopsis (Arabidopsis thaliana) contain a large vegetative cell and two smaller sperm cells. Pollen grains express AtTIP1;3 and AtTIP5;1, two members of the Tonoplast Intrinsic Protein subfamily of aquaporins. To address the spatial and temporal expression pattern of the two homologs, C-terminal fusions of AtTIP1;3 and AtTIP5;1 with green fluorescent protein and mCherry, respectively, were expressed in transgenic Arabidopsis under the control of their native promoter. Confocal laser scanning microscopy revealed that AtTIP1;3 and AtTIP5;1 are specific for the vacuoles of the vegetative and sperm cells, respectively. The tonoplast localization of AtTIP5;1 was established by reference to fluorescent protein markers for the mitochondria and vacuoles of sperm and vegetative cells and is at variance with the claim that AtTIP5;1 is localized in vegetative cell mitochondria. AtTIP1;3-green fluorescent protein and AtTIP5;1-mCherry showed concomitant expression, from first pollen mitosis up to pollen tube penetration in the ovule, thereby revealing the dynamics of vacuole morphology in maturating and germinating pollen. Transfer DNA insertion mutants for either AtTIP1;3 or AtTIP5;1 showed no apparent growth phenotype and had no significant defect in male transmission of the mutated alleles. By contrast, a double knockout displayed an abnormal rate of barren siliques, this phenotype being more pronounced under limited water or nutrient supply. The overall data indicate that vacuoles of vegetative and sperm cells functionally interact and contribute to male fertility in adverse environmental conditions.

  9. Apical blebs on sperm-storage tubule epithelial cell microvilli: their release and interaction with resident sperm in the turkey hen oviduct

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Technical Abstract: Located at the anterior end of the turkey hen vagina are numerous discrete tubular invaginations of the surface epithelium, collectively referred to as the sperm-storage tubules (SSTs). Following mating or artificial insemination, sperm ascend the vagina, enter the SSTs, and ove...

  10. Sperm Cell Population Dynamics in Ram Semen during the Cryopreservation Process

    PubMed Central

    Ramón, Manuel; Pérez-Guzmán, M. Dolores; Jiménez-Rabadán, Pilar; Esteso, Milagros C.; García-Álvarez, Olga; Maroto-Morales, Alejandro; Anel-López, Luis; Soler, Ana J.; Fernández-Santos, M. Rocío; Garde, J. Julián

    2013-01-01

    Background Sperm cryopreservation has become an indispensable tool in biology. Initially, studies were aimed towards the development of efficient freezing protocols in different species that would allow for an efficient storage of semen samples for long periods of time, ensuring its viability. Nowadays, it is widely known that an important individual component exists in the cryoresistance of semen, and efforts are aimed at identifying those sperm characteristics that may allow us to predict this cryoresistance. This knowledge would lead, ultimately, to the design of optimized freezing protocols for the sperm characteristics of each male. Methodology/Principal Findings We have evaluated the changes that occur in the sperm head dimensions throughout the cryopreservation process. We have found three different patterns of response, each of one related to a different sperm quality at thawing. We have been able to characterize males based on these patterns. For each male, its pattern remained constant among different ejaculates. This latter would imply that males always respond in the same way to freezing, giving even more importance to this sperm feature. Conclusions/Significance Changes in the sperm head during cryopreservation process have resulted useful to identify the ability of semen of males for freezing. We suggest that analyses of these response patterns would represent an important tool to characterize the cryoresistance of males when implemented within breeding programs. We also propose follow-up experiments to examine the outcomes of the use of different freezing protocols depending on the pattern of response of males. PMID:23544054

  11. DNA (deoxyribonucleic acid) synthesis following microinjection of heterologous sperm and somatic cell nuclei into hamster oocytes

    SciTech Connect

    Naish, S.J.; Perreault, S.D.; Zirkin, B.R.

    1987-01-01

    The authors investigated the ability of the hamster oocyte to initiate DNA synthesis in nuclei differing in basic protein content. DNA synthesis was studied by autoradiography in oocytes that had been incubated in /sup 3/H-thymidine after being parthenogenetically activated by sham microinjection, or microinjected with hamster, mouse, rabbit, or fish sperm nuclei, or hamster hepatocyte nuclei. Within 6 hr of sham or nucleus microinjection, nuclei of each type underwent transformation into pronuclei and synthesized DNA. These results demonstrated that the hamster egg can access and utilize its own and each type of template provided, whether homologous or heterologous. However, pronuclei derived from hamster sperm nuclei were more likely to be synthesizing DNA at 6 hr than pronuclei derived from sperm nuclei of other species. The authors conclude that the mechanisms employed by the hamster oocyte to transform hamster sperm nuclei into pronuclei and to effect DNA synthesis in these nuclei are not specific for the hamster sperm nucleus. Nevertheless, these mechanisms apparently operate more efficiently when the hamster sperm nucleus, rather than a heterologous sperm nucleus, is present.

  12. Absence of beneficial effects on rabbit sperm cell cryopreservation by several antioxidant agents.

    PubMed

    Maya-Soriano, M J; Taberner, E; Sabés-Alsina, M; Piles, M; Lopez-Bejar, M

    2015-02-01

    The generation of reactive oxygen species associated with cryopreservation could be responsible for mammalian sperm damage and the limitable value of stored semen in artificial insemination. The aim of this study was to assess several antioxidant agents supplemented in a commercial freezing extender (Gent B®) in order to improve post-thaw rabbit sperm quality. Ejaculates of 26 New Zealand White rabbit bucks were collected, evaluated and frozen using a conventional protocol. Antioxidant agents were tested at different concentrations: bovine serum albumin (BSA; 5, 30 or 60 mg/ml), retinol (RO; 50, 100 or 200 μM) and retinyl (RI; 0.282 or 2.82 μg/ml). Per cent viability, morphological abnormalities and intact acrosomes were determined using eosin-nigrosin staining. Motility and progressivity were analyzed by computer-assisted sperm analysis (CASA). In general, all sperm quality parameters were negatively affected by the cryopreservation process, the largest effect seen was for total motility. The addition of antioxidant agents did not improve thaw sperm quality. Furthermore, for RI groups a significant decrease in sperm quality parameters was recorded. In conclusion, rabbit sperm quality is negatively affected by the cryopreservation process. To our knowledge this report is the first using these antioxidants to supplement rabbit freezing extender. BSA and RO at concentrations used in the study did not improve sperm quality parameters after thawing, whereas RI supplementation appeared to be toxic. More studies are required to find the appropriate antioxidants necessary and their most effective concentrations to improve rabbit post-thaw sperm quality.

  13. Morphological diversity of sperm: A mini review

    PubMed Central

    Prakash, Seppan; Prithiviraj, Elumalai; Suresh, Sekar; Lakshmi, Nagella Venkata; Ganesh, Mohanraj Karthik; Anuradha, Murugesan; Ganesh, Lakshmanan; Dinesh, Premavathy

    2014-01-01

    Sperms are highly specialized cells for delivering DNA from male to the ovum. Incredibly, wide degree of diversity in sperm morphology in their basic structures i.e. head, middle piece and tail is found across species. Differences in terms of overall size of the sperm, shape and number of sperm produced are also incredible. One of the key for this variations or diversity in sperm may be associated with female reproductive tract, sperm competition, testicular size and sperm size and number. Establishing a correlation between sperm morphology and factors influencing them is a phenomenal task. In this mini-review these associations and the anatomical and functional adaptations among different from of sperm cells that have evolved to optimize fertilization success are discussed. Nevertheless, explaining these morphological diversities in sperm cells is a challenging question and it seems that evolutionary biologists have only recently engaged in exploring its links and patterns. From the literatures it seems that there is no causal relationship between sperm size and testicular size, however, the accumulated knowledge do indicates evolution of sperm morphology across species has some associations with female reproductive tract, sperm competition and sperm size and number, however interpreting these results for phylogentic correlations should be approached with caution. PMID:24976817

  14. B-cell lymphoma-2 localization in the female reproductive tract of the Chinese soft-shelled turtle, Pelodiscus sinensis and its relationship with sperm storage.

    PubMed

    Le, Yuan; Chen, Shaofan; Hu, Lisi; Zhang, Linli; Ullah, Shakeeb; Liu, Tengfei; Yang, Ping; Liu, Yi; Chen, Qiusheng

    2015-12-01

    The aim of the present study was to investigate the expression and localization of B-cell lymphoma-2 (Bcl-2) in the oviduct of the Chinese soft-shelled turtle, Pelodiscus sinensis, during the reproductive cycle to analyze the relationship between Bcl-2 and sperm storage. Bcl-2 expression was confirmed in the P. sinensis oviduct by western blot analysis. Hematoxylin-eosin staining showed that female P. sinensis stored sperm from November to April of the following year. The oviduct showed positive immunostaining for Bcl-2 of epithelial ciliated cells, gland ducts, and gland cells. Bcl-2 expression in the oviduct was associated with sperm storage occurrence. This indicates that the survival factor Bcl-2 may play a role in P. sinensis sperm storage.

  15. A Procedure-Spanning Analysis of Plasma Membrane Integrity for Assessment of Cell Viability in Sperm Cryopreservation of Zebrafish Danio rerio.

    PubMed

    Yang, Huiping; Daly, Jonathan; Carmichael, Carrie; Matthews, Jen; Varga, Zoltan M; Tiersch, Terrence

    2016-04-01

    The goal of this study was to evaluate plasma membrane integrity and motility for zebrafish sperm quality assessment along the cryopreservation pathway-from sample collection through refrigerated storage, cryoprotectant equilibration, freezing, thawing, and fertilization. The objectives were to: (1) evaluate the effects of osmolality, extender, and refrigerated storage on sperm plasma membrane integrity and motility, and (2) compare cryopreservation of sperm from farm-raised and well-characterized research populations by evaluating motility and membrane integrity of fresh, post-equilibration (before freezing) and post-thaw sperm, and post-thaw fertility. Osmolality, extender, and storage time each influenced sperm motility and membrane integrity. Isotonic osmolality showed the best protection for motility and membrane integrity compared to hypotonic and hypertonic osmolalities. Of the four tested extenders, Hanks' balanced salt solution (HBSS) and Ca(2+)-free HBSS showed the best protection compared with NaCl and glucose, and sperm retained motility and membrane integrity for 24 h of refrigerated storage. Sperm cryopreservation of zebrafish from a farm population (n = 20) and an AB research line (n = 20) showed significant differences in post-thaw fertility (32% ± 18% vs. 73% ± 21%). No differences were found in post-thaw motility, although the farm-raised zebrafish possessed a larger body size, testis weight, and higher fresh motility. Correlation analysis of pooled data did not identify correlations among motility, flow cytometry analysis of membrane integrity and recognizable cells, and post-thaw sperm fertility (p ≥ 0.202). More research is needed to standardize the fertilization conditions especially sperm-to-egg ratio to avoid possible overabundance of sperm to obscure the differences.

  16. Characterization of epididymal sperm motion and its correlation with stages of target cells in rats given alpha-chlorohydrin, cyclophosphamide or nitrazepam.

    PubMed

    Kaneto, M; Kanamori, S; Hara, K; Kishi, K

    1999-08-01

    Epididymal sperm motion in rats was characterized by computer-aided sperm motion analysis (CASA) with its correlation to testicular lesions in the 2-week treatment study, using three compounds which are known to affect different stages of germ cells. Mature male rats were treated daily for 2 weeks with alpha-chlorohydrin (alpha-CH, 5 mg/kg), cyclophosphamide (CP, 20 mg/kg) or nitrazepam (NZ, 20, 40, 60 mg/kg). Changes in sperm motion were detected only in the alpha-CH and 60-mg/kg NZ-treated groups. Of the sperm motion parameters, velocity and amplitude of lateral head displacement (ALH) were concomitantly reduced in these two groups with good correlation. With respect to the distribution of the values in parameters, however, alpha-CH shifted the values down within a small range with high percentages of motile sperm, while NZ distributed them over a wide range with low percentages of motile sperm. CP treatment showed no histopathological changes in advanced germ cells, though it showed a decrease in the number of early germ cells. NZ treatment affected round and elongating spermatids (approximately step 14) at doses of 20 and 40 mg/kg, and affected also more advanced spermatids (approximately step 19) at the dose of 60 mg/kg. alpha-CH treatment did not affect testicular histopathology. These findings indicate that 60-mg/kg NZ treatment reduced sperm motion as a result of lesions affected in elongated spermatids and alpha-CH reduced it by direct effects on epididymal spermatozoa. The present study indicates that in addition to percentage of motile sperm, the velocity and ALH can be useful to detect the changes in sperm motion caused by different actions of NZ and alpha-CH, though each compound showed a distinct distribution pattern of these parameters.

  17. Tuning sperm chemotaxis.

    PubMed

    Guerrero, Adán; Wood, Christopher D; Nishigaki, Takuya; Carneiro, Jorge; Darszon, Alberto

    2010-10-01

    Sperm chemotaxis is a long-term puzzle and most of our knowledge comes from studying marine animals that are external fertilizers. Sperm are attracted by diffusible chemical factors (chemoattractants) released from the egg which redirect their swimming paths towards their source. This redirection is driven by increases in flagellar curvature that correlate with transient flagellar Ca(2+) increases. Recent experimental and modelling results provide insights into the signal flow underlying the translation of an external chemical gradient into an intracellular molecular and motor response. A fundamental element of sea-urchin sperm chemotaxis lies in the ability of these cells to suppress Ca(2+)-mediated increases in flagellar curvature while experiencing an increasing chemoattractant gradient. The article considers this new evidence and summarizes the known underlying cellular mechanisms and behavioural strategies that sperm use to locate and fertilize the oocyte.

  18. Comparative cytotoxicity and genotoxicity of particulate and soluble hexavalent chromium in human and sperm whale (Physeter macrocephalus) skin cells.

    PubMed

    Li Chen, Tânia; LaCerte, Carolyne; Wise, Sandra S; Holmes, Amie; Martino, Julieta; Wise, John Pierce; Thompson, W Douglas; Wise, John Pierce

    2012-01-01

    Chromium (Cr) is a global marine pollutant, present in marine mammal tissues. Hexavalent chromium [Cr(VI)] is a known human carcinogen. In this study, we compare the cytotoxic and clastogenic effects of Cr(VI) in human (Homo sapiens) and sperm whale (Physeter macrocephalus) skin fibroblasts. Our data show that increasing concentrations of both particulate and soluble Cr(VI) induce increasing amounts of cytotoxicity and clastogenicity in human and sperm whale skin cells. Furthermore, the data show that sperm whale cells are resistant to these effects exhibiting less cytotoxicity and genotoxicity than the human cells. Differences in Cr uptake accounted for some but not all of the differences in particulate and soluble Cr(VI) genotoxicity, although it did explain the differences in particulate Cr(VI) cytotoxicity. Altogether, the data indicate that Cr(VI) is a genotoxic threat to whales, but also suggest that whales have evolved cellular mechanisms to protect them against the genotoxicity of environmental agents such as Cr(VI).

  19. Subcellular Localization, Stability, and trans-Cleavage Competence of the Hepatitis C Virus NS3-NS4A Complex Expressed in Tetracycline-Regulated Cell Lines

    PubMed Central

    Wölk, Benno; Sansonno, Domenico; Kräusslich, Hans-Georg; Dammacco, Franco; Rice, Charles M.; Blum, Hubert E.; Moradpour, Darius

    2000-01-01

    A tetracycline-regulated gene expression system and a panel of novel monoclonal antibodies were used to examine the subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus (HCV) NS3-NS4A complex in inducible cell lines. The NS3 serine protease domain and the full-length NS3 protein expressed in the absence of the NS4A cofactor were diffusely distributed in the cytoplasm and nucleus. Coexpression of NS4A, however, directed NS3 to the endoplasmic reticulum (ER) or an ER-like modified compartment, as demonstrated by colocalization with 3,3′-dihexyloxacarbocyanine iodide, protein disulfide isomerase, and calnexin, as well as subcellular fractionation analyses. In addition, coexpression with NS4A dramatically increased the intracellular stability of NS3 (mean protein half-life of 26 versus 3 h) and allowed for NS4A-dependent trans-cleavage at the NS4B-NS5A junction. Deletion analyses revealed that the hydrophobic amino-terminal domain of NS4A was required for ER targeting of NS3. These results demonstrate the importance of studying HCV proteins in their biological context and define a well-characterized cell culture system for further analyses of the NS3-NS4A complex and the evaluation of novel antiviral strategies against hepatitis C. PMID:10666260

  20. Bile acids alter the subcellular localization of CNT2 (concentrative nucleoside cotransporter) and increase CNT2-related transport activity in liver parenchymal cells

    PubMed Central

    Fernández-Veledo, Sonia; Huber-Ruano, Isabel; Aymerich, Ivette; Duflot, Sylvie; Casado, F. Javier; Pastor-Anglada, Marçal

    2006-01-01

    CNT2 (concentrative nucleoside cotransporter) is a plasma membrane high-affinity Na+-coupled adenosine transporter, also localized in intracellular structures. This transporter protein may play additional roles other than nucleoside salvage, since it has recently been shown to be under purinergic control via KATP channels, by a mechanism that does not seem to involve changes in its subcellular localization. In an attempt to identify the agents that promote CNT2 trafficking, bile acids were found to increase CNT2-related transport activity in a KATP channel-independent manner in both Fao hepatoma and rat liver parenchymal cells. A maximum effect was recorded after treatment with hydrophilic anions such as TCA (taurocholate). However, this effect did not involve changes in the amount of CNT2 protein, it was instead associated with a subcellular redistribution of CNT2, resulting in an accumulation of the transporter at the plasma membrane. This was deduced from subcellular fractionation studies, biotinylation of plasma membrane proteins and subsequent CNT2 detection in streptavidin precipitates and in vivo confocal microscopic analysis of the distribution of a YFP (yellow fluorescent protein)–CNT2 construct. The induction of CNT2 translocation, triggered by TCA, was inhibited by wortmannin, dibutyryl-AMPc, PD98059 and colchicine, thus suggesting the involvement of the PI3K/ERK (phosphoinositide 3-kinase/extracellular-signal related kinase) pathway in microtubule-dependent activation of recombinant CNT2. These are novel effects of bile-acid physiology and provide the first evidence for short-term regulation of CNT2 translocation into and from the plasma membrane. PMID:16390326

  1. Three-dimensional mid-infrared tomographic imaging of endogenous and exogenous molecules in a single intact cell with subcellular resolution.

    PubMed

    Quaroni, Luca; Obst, Martin; Nowak, Marcus; Zobi, Fabio

    2015-01-02

    Microscopy in the mid-infrared spectral range provides detailed chemical information on a sample at moderate spatial resolution and is being used increasingly in the characterization of biological entities as challenging as single cells. However, a conventional cellular 2D imaging measurement is limited in its ability to associate specific compositional information to subcellular structures because of the interference from the complex topography of the sample. Herein we provide a method and protocols that overcome this challenge in which tilt-series infrared tomography is used with a standard benchtop infrared microscope. This approach gives access to the quantitative 3D distribution of molecular components based on the intrinsic contrast provided by the sample. We demonstrate the method by quantifying the distribution of an exogenous metal carbonyl complex throughout the cell and by reporting changes in its coordination sphere in different locations in the cell.

  2. Subcellular localization of pituitary enzymes

    NASA Technical Reports Server (NTRS)

    Smith, R. E.

    1970-01-01

    A cytochemical procedure is reported for identifying subcellular sites of enzymes hydrolyzing beta-naphthylamine substrates, and to study the sites of reaction product localization in cells of various tissues. Investigations using the substrate Leu 4-methoxy-8-naphthylamine, a capture with hexonium pararosaniline, and the final chelation of osmium have identified the hydrolyzing enzyme of rat liver cells; this enzyme localized on cell membranes with intense deposition in the areas of the parcanaliculi. The study of cells in the anterior pituitary of the rat showed the deposition of reaction product on cell membrane; and on the membranes of secretion granules contained within the cell. The deposition of reaction product on the cell membrane however showed no increase or decrease with changes in the physiological state of the gland and release of secretion granules from specific cells.

  3. [From spermatic animalcules to sperm cells: the reconceptualization of generation in the 19th century].

    PubMed

    Vienne, Florence

    2009-09-01

    At the end of the 18th and still at the beginning of the 19th century most naturalists considered spermatic animalcules to be parasites of the seminal fluid that played no role in procreation. This view was progressively questioned by 19th century physiologists. They gradually redefined the spermatic animals as (cellular) products of the male organism, as agents of fertilization and bearers of the male heredity material. This article discusses this change from two different perspectives: on a microhistorical level, it analyzes the experimental research of the naturalist Lazzaro Spallanzani (1729-1799) and of the physiologist Albert Kölliker (1817-1905) in order to show how spermatozoa were turned into a new epistemic object of biology--the sperm cell. Further, it asks how the role of the reconceptualization of spermatic animalcules affected the long-term transformations that gave rise of our modern understanding of heredity, generation and the sexed body. By combining these two perspectives, the article aims to connect historiographies that are often kept separate: the macrohistorical narratives about gender and the body in the modern age and the microhistorical studies of biomedical practices and objects.

  4. Relative testis size and sperm morphometry across mammals: no evidence for an association between sperm competition and sperm length.

    PubMed Central

    Gage, Matthew J G; Freckleton, Robert P

    2003-01-01

    Understanding why there is extensive variation in sperm form and function across taxa has been a challenge because sperm are specialized cells operating at a microscopic level in a complex environment. This comparative study collates published data to determine whether the evolution of sperm morphometry (sperm total length and separate component dimensions) is associated with sperm competition (when different males' sperm mix and compete for a female's ova) across 83 mammalian species. We use relative testes mass as an indicator of the intensity of sperm competition across taxa: relative investment into testes is widely accepted to predict the level of sperm competition that a species or population endures. Although we found evidence for positive associations between relative testes mass (controlling for allometry) and sperm morphometry across 83 mammalian species, these relationships were phylogenetically dependent. When we appropriately controlled for phylogenetic association using multiple regression within a phylogenetic framework, there was no relationship between relative testes mass and sperm length across mammals. Furthermore, we found no evidence for associations between relative testes mass and sperm head, mid-piece or flagellar lengths, nor was there a relationship with mid-piece or mitochondrial volumes. Results, therefore, indicate that sperm competition does not select for longer or shorter sperm across mammals, and alternative forces selecting on sperm form and function are discussed. PMID:12769463

  5. No evidence of sperm conjugate formation in an Australian mouse bearing sperm with three hooks

    PubMed Central

    Firman, Renée C; Bentley, Blair; Bowman, Faye; Marchant, Fernando García-Solís; Parthenay, Jahmila; Sawyer, Jessica; Stewart, Tom; O'Shea, James E

    2013-01-01

    Sperm conjugation occurs when two or more sperm physically unite for motility or transport through the female reproductive tract. In many muroid rodent species, sperm conjugates have been shown to form by a single, conspicuous apical hook located on the sperm head. These sperm “trains” have been reported to be highly variable in size and, despite all the heads pointing in roughly the same direction, exhibit a relatively disordered arrangement. In some species, sperm “trains” have been shown to enhance sperm swimming speed, and thus have been suggested to be advantageous in sperm competition. Here, we assessed the behavior of sperm in the sandy inland mouse (Pseudomys hermannsburgensis), a muroid rodent that bears sperm with three apical hooks. First, we accrued genetic evidence of multiple paternity within “wild” litters to unequivocally show that sperm competition does occur in this species. Following this we utilized both in vitro and in vivo methodologies to determine whether sandy inland mouse sperm conjugate to form motile trains. Our observations of in vitro preparations of active sperm revealed that sandy inland mouse sperm exhibit rapid, progressive motility as individual cells only. Similarly, histological sections of the reproductive tracts of mated females revealed no in vivo evidence of sperm conjugate formation. We conclude that the unique, three-hooked morphology of the sandy inland mouse sperm does not facilitate the formation of motile conjugates, and discuss our findings in relation to the different hypotheses for the evolution of the muroid rodent hook/s. PMID:23919134

  6. Characterization of pig sperm hyaluronidase and improvement of the digestibility of cumulus cell mass by recombinant pSPAM1 hyaluronidase in an in vitro fertilization assay.

    PubMed

    Yoon, Sungwon; Chang, Kyu-Tae; Cho, Hongsang; Moon, Jisang; Kim, Ju-Sung; Min, Sung-Hun; Koo, Deog-Bon; Lee, Sang-Rae; Kim, Sang-Hyun; Park, Ki-Eun; Park, Young Il; Kim, Ekyune

    2014-11-30

    Although sperm hyaluronidase is thought to play an important role in mammalian fertilization, the molecular function underlying these steps remains largely unknown. In mouse models, sperm-specific SPAM1 and HYAL5 hyaluronidase are believed to function in both sperm penetration of the cumulus matrix and sperm-ZP binding. However, gene-targeting studies for SPAM1 or HYAL5 show that hyaluronidases are not essential for fertilization, despite the fact that exogenous hyaluronidase can disrupt the cumulus matrix. Therefore, to evaluate whether sperm hyaluronidase is essential for mammalian fertilization, it is necessary to generate HYAL5/SPAM1 double-knockout mice. However, generating double-knockout mice is very difficult because these two genes exist on the same chromosome. Recently, investigators have begun to employ the pig model system to study human disease due to its similarities to human anatomy and physiology. In this study, we confirmed that pig SPAM1 exists as a single copy gene on chromosome 18 and is specifically expressed in the testis. In addition, we expressed recombinant pig SPAM1 in human embryonic kidney 293 cells and showed that these enzymes possess hyaluronidase activity. We also demonstrated that a polyclonal antibody against pig sperm hyaluronidase inhibits sperm-egg interactions in an in vitro fertilization (IVF) assay. Our results suggest that pig SPAM1 may play a critical role in pig fertilization and that recombinant SPAM1 can disperse the oocyte-cumulus complex in an IVF assay.

  7. Sperm dynamics in spiders (Araneae): ultrastructural analysis of the sperm activation process in the garden spider Argiope bruennichi (Scopoli, 1772).

    PubMed

    Vöcking, Oliver; Uhl, Gabriele; Michalik, Peter

    2013-01-01

    Storage of sperm inside the female genital tract is an integral phase of reproduction in many animal species. The sperm storage site constitutes the arena for sperm activation, sperm competition and female sperm choice. Consequently, to understand animal mating systems information on the processes that occur from sperm transfer to fertilization is required. Here, we focus on sperm activation in spiders. Male spiders produce sperm whose cell components are coiled within the sperm cell and that are surrounded by a proteinaceous sheath. These inactive and encapsulated sperm are transferred to the female spermathecae where they are stored for later fertilization. We analyzed the ultrastructural changes of sperm cells during residency time in the female genital system of the orb-web spider Argiope bruennichi. We found three clearly distinguishable sperm conditions: encapsulated sperm (secretion sheath present), decapsulated (secretion sheath absent) and uncoiled sperm (cell components uncoiled, presumably activated). After insemination, sperm remain in the encapsulated condition for several days and become decapsulated after variable periods of time. A variable portion of the decapsulated sperm transforms rapidly to the uncoiled condition resulting in a simultaneous occurrence of decapsulated and uncoiled sperm. After oviposition, only decapsulated and uncoiled sperm are left in the spermathecae, strongly suggesting that the activation process is not reversible. Furthermore, we found four different types of secretion in the spermathecae which might play a role in the decapsulation and activation process.

  8. Calpain-mediated Processing of p53-associated Parkin-like Cytoplasmic Protein (PARC) Affects Chemosensitivity of Human Ovarian Cancer Cells by Promoting p53 Subcellular Trafficking*

    PubMed Central

    Woo, Michael G.; Xue, Kai; Liu, Jiayin; McBride, Heidi; Tsang, Benjamin K.

    2012-01-01

    Resistance to cisplatin (CDDP)-based therapy is a major hurdle to the successful treatment of human ovarian cancer (OVCA), and the chemoresistant phenotype in OVCA cells is associated with Akt-attenuated p53-mediated apoptosis. Pro-apoptotic functions of p53 involve both transcription-dependent and -independent signaling pathways, and dysfunctional localization and/or inactivation of p53 contribute to the development of chemoresistance. PARC is a cytoplasmic protein regulating p53 subcellular localization and subsequent function. Little is known about the molecular mechanisms regulating PARC. Although PARC contains putative caspase-3 cleavage sites, and CDDP is known to induce the activation of caspases and calpains and induce proteasomal degradation of anti-apoptotic proteins, if and how PARC is regulated by CDDP in OVCA are unknown. Here, we present evidence that CDDP promotes calpain-mediated PARC down-regulation, mitochondrial and nuclear p53 accumulation, and apoptosis in chemosensitive but not resistant OVCA cells. Inhibition of Akt is required to sensitize chemoresistant cells to CDDP in a p53-dependent manner, an effect enhanced by PARC down-regulation. CDDP-induced PARC down-regulation is reversible by inhibition of calpain but not of caspases or the 26 S proteasome. Furthermore, in vitro experiments confirm the ability of calpain in mediating Ca2+-dependent PARC down-regulation. The role of Ca2+ in PARC down-regulation was further confirmed as ionomycin-induced PARC down-regulation in both chemosensitive and chemoresistant ovarian cancer cells. The data presented here implicate the regulation of p53 subcellular localization and apoptosis by PARC as a contributing factor in CDDP resistance in OVCA cells and Ca2+/calpain in PARC post-translational processing and chemosensitivity. PMID:22117079

  9. Static magnetic fields inhibit proliferation and disperse subcellular localization of gamma complex protein3 in cultured C2C12 myoblast cells.

    PubMed

    Kim, SeungChan; Im, Wooseok

    2010-05-01

    Magnetic fields may delay the rate of cell cycle progression, and there are reports that magnetic fields induce neurite outgrowth in cultured neuronal cells. To demonstrate whether magnetic field also effects on myoblast cells in cell growth, C2C12 cell lines were cultured and 2000G static magnetic field was applied. After 48 h of incubation, both the WST-1 assay (0.01 < P < 0.025, t-test) and direct cell counting (P < 0.0005, t-test) showed that static magnetic fields inhibit the proliferation of cultured C2C12 cells. Immunocytochemistry for alpha and tubulin gamma complex protein (TUBA and GCP3) was made and applying a static magnetic field-dispersed tubulin GCP3 formation, a intracellular apparatus for tubulin structuring in cell division. This protein expression was not altered by western blot. This study indicates that applying a static magnetic field alters the subcellular localizing of GCP3, and may delay the cell growth in cultured C2C12 myoblast cells.

  10. Antiproliferative effect of dihydroxyacetone on Trypanosoma brucei bloodstream forms: cell cycle progression, subcellular alterations, and cell death.

    PubMed

    Uzcátegui, Néstor L; Carmona-Gutiérrez, Didac; Denninger, Viola; Schoenfeld, Caroline; Lang, Florian; Figarella, Katherine; Duszenko, Michael

    2007-11-01

    We evaluated the effects of dihydroxyacetone (DHA) on Trypanosoma brucei bloodstream forms. DHA is considered an energy source for many different cell types. T. brucei takes up DHA readily due to the presence of aquaglyceroporins. However, the parasite is unable to use it as a carbon source because of the absence of DHA kinase (DHAK). We could not find a homolog of the relevant gene in the genomic database of T. brucei and have been unable to detect DHAK activity in cell lysates of the parasite, and the parasite died quickly if DHA was the sole energy source in the medium. In addition, during trypanosome cultivation, DHA induced growth inhibition with a 50% inhibitory concentration of about 1 mM, a concentration that is completely innocuous to mammals. DHA caused cell cycle arrest in the G(2)/M phase of up to 70% at a concentration of 2 mM. Also, DHA-treated parasites showed profound ultrastructural alterations, including an increase of vesicular structures within the cytosol and the presence of multivesicular bodies, myelin-like structures, and autophagy-like vacuoles, as well as a marked disorder of the characteristic mitochondrion structure. Based on the toxicity of DHA for trypanosomes compared with mammals, we consider DHA a starting point for a rational design of new trypanocidal drugs.

  11. Ultrastructure and lipid composition of detergent-resistant membranes derived from mammalian sperm and two types of epithelial cells.

    PubMed

    van Gestel, Renske A; Brouwers, Jos F; Ultee, Anton; Helms, J Bernd; Gadella, Bart M

    2016-01-01

    Lipid rafts are micro-domains of ordered lipids (Lo phase) in biological membranes. The Lo phase of cellular membranes can be isolated from disordered lipids (Ld phase) after treatment with 1 % Triton  X-100 at 4 °C in which the Lo phase forms the detergent-resistant membrane (DRM) fraction. The lipid composition of DRM derived from Madin-Darby canine kidney (MDCK) cells, McArdle cells and porcine sperm is compared with that of the whole cell. Remarkably, the unsaturation and chain length degree of aliphatic chains attached to phospholipids is virtually the same between DRM and whole cells. Cholesterol and sphingomyelin were enriched in DRMs but to a cell-specific molar ratio. Sulfatides (sphingolipids from MDCK cells) were enriched in the DRM while a seminolipid (an alkylacylglycerolipid from sperm) was depleted from the DRM. Treatment with <5 mM methyl-ß-cyclodextrin (MBCD) caused cholesterol removal from the DRM without affecting the composition and amount of the phospholipid while higher levels disrupted the DRM. The substantial amount of (poly)unsaturated phospholipids in DRMs as well as a low stoichiometric amount of cholesterol suggest that lipid rafts in biological membranes are more fluid and dynamic than previously anticipated. Using negative staining, ultrastructural features of DRM were monitored and in all three cell types the DRMs appeared as multi-lamellar vesicular structures with a similar morphology. The detergent resistance is a result of protein-cholesterol and sphingolipid interactions allowing a relatively passive attraction of phospholipids to maintain the Lo phase. For this special issue, the relevance of our findings is discussed in a sperm physiological context.

  12. The use of complimentary assays to evaluate the enrichment of human sperm quality in asthenoteratozoospermic and teratozoospermic samples processed with Annexin-V magnetic activated cell sorting.

    PubMed

    Delbes, G; Herrero, M B; Troeung, E-T; Chan, P T K

    2013-09-01

    Sperm chromatin integrity may affect the outcomes of assisted reproductive technology (ART). Developing a clinically reliable strategy to enrich sperm samples with high chromatin quality spermatozoa prior to sperm banking or use in ART would thus be advantageous. The objectives of this study were to: (i) assess the sperm chromatin quality in men with different categories of semen parameters; and (ii) evaluate the extents of Annexin-V magnetic-activated cell sorting (MACS) technology coupled with differential density gradient centrifugation (DGC) in improving sperm chromatin quality. Three categories of men from couples attending a university-based fertility clinic were recruited based on their semen parameters: normozoospermic (n = 13), asthenoteratozoospermic (n = 17) and teratozoospermic (n = 12). For each patient, spermatozoa in semen samples were processed first by DGC to enrich the motility and further by MACS to remove spermatozoa showing apoptotic features. The yield and enrichment of sperm quality was evaluated at each step with conventional semen parameters in conjunction with a combination of five complementary assays, to assess sperm maturity, chromatin structure, compaction and DNA integrity (Hyaluronic Binding Assay, SCSA, chromomycine A3 staining and TUNEL and COMET assays). Our results demonstrated that, compared with normozoospermic samples, raw asthenoteratozoospermic and teratozoospermic samples had a higher proportion of spermatozoa containing DNA breaks, but only asthenoteratozoospermic exhibited altered chromatin structure and decreased binding to hyaluronic acid. Interestingly, the DGC appeared to select for more mature spermatozoa with high DNA compaction. More importantly, in all categories of semen samples, Annexin-V MACS allows enrichment of spermatozoa with good chromatin quality as measured by the TUNEL and SCSA. Because effective treatment modalities to improve sperm DNA damage are limited, our results suggest a potential clinical

  13. Human sperm acrosome reaction-initiating activity associated with the human cumulus oophorus and mural granulosa cells.

    PubMed

    Siiteri, J E; Dandekar, P; Meizel, S

    1988-04-01

    This report describes the detection and partial characterization of preovulatory human cumulus oophorus and mural granulosa cell-associated activity capable of initiating the human sperm acrosome reaction (AR) in vitro. Fragments of preovulatory human cumulus (cells plus extracellular matrix) were washed 3 times, incubated for 24 hr and the spent media and washes assayed for their ability to initiate the human sperm acrosome reaction (AR) in vitro. AR activity was present in the first two washes but not the third wash; however, AR activity was recovered in the spent medium after 3 X-washed fragments were incubated for 24 hr under conditions which maintained the viability of the cumulus cells. The spent media of preovulatory human mural granulosa cells contained AR-initiating activity after 1-3, 3-6, and 6-9 days of culture. The properties of the AR activity present in spent media of human cumulus fragments included resistance to loss of activity during treatment with pronase; resistance to loss of activity during treatment with chondroitinase ABC or bacterial hyaluronidase; heat stability after overnight incubation; lack of extraction by chloroform-methanol; an apparent molecular weight (MW) of 50,000, as determined by Sephadex G-75 column chromatography; conversion to a lower apparent MW activity by incubation with pronase. These properties are also characteristic of a fraction derived by Sephadex G-75 chromatography of preovulatory human follicular fluid which also has been shown to stimulate the human sperm acrosome reaction in vitro. The AR activity from spent media of human mural granulosa cells is also found in a 50,000 MW Sephadex G-75 fraction. We propose that the sources of the 50,000 MW human follicular fluid AR activity are the cumulus oophorus and the mural granulosa cells.

  14. Autophagosome Proteins LC3A, LC3B and LC3C Have Distinct Subcellular Distribution Kinetics and Expression in Cancer Cell Lines

    PubMed Central

    Koukourakis, Michael I.; Kalamida, Dimitra; Giatromanolaki, Alexandra; Zois, Christos E.; Sivridis, Efthimios; Pouliliou, Stamatia; Mitrakas, Achilleas; Gatter, Kevin C.; Harris, Adrian L.

    2015-01-01

    LC3s (MAP1-LC3A, B and C) are structural proteins of autophagosomal membranes, widely used as biomarkers of autophagy. Whether these three LC3 proteins have a similar biological role in autophagy remains obscure. We examine in parallel the subcellular expression patterns of the three LC3 proteins in a panel of human cancer cell lines, as well as in normal MRC5 fibroblasts and HUVEC, using confocal microscopy and western blot analysis of cell fractions. In the cytoplasm, there was a minimal co-localization between LC3A, B and C staining, suggesting that the relevant autophagosomes are formed by only one out of the three LC3 proteins. LC3A showed a perinuclear and nuclear localization, while LC3B was equally distributed throughout the cytoplasm and localized in the nucleolar regions. LC3C was located in the cytoplasm and strongly in the nuclei (excluding nucleoli), where it extensively co-localized with the LC3A and the Beclin-1 autophagy initiating protein. Beclin 1 is known to contain a nuclear trafficking signal. Blocking nuclear export function by Leptomycin B resulted in nuclear accumulation of all LC3 and Beclin-1 proteins, while Ivermectin that blocks nuclear import showed reduction of accumulation, but not in all cell lines. Since endogenous LC3 proteins are used as major markers of autophagy in clinical studies and cell lines, it is essential to check the specificity of the antibodies used, as the kinetics of these molecules are not identical and may have distinct biological roles. The distinct subcellular expression patterns of LC3s provide a basis for further studies. PMID:26378792

  15. The presence of centrioles and centrosomes in ovarian mature cystic teratoma cells suggests human parthenotes developed in vitro can differentiate into mature cells without a sperm centriole

    SciTech Connect

    Lee, Bo Yon; Shim, Sang Woo; Kim, Young Sun; Kim, Seung Bo

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer The sperm centriole is the progenitor of centrosomes in all somatic cells. Black-Right-Pointing-Pointer Centrioles and centrosomes exist in parthenogenetic ovarian teratoma cells. Black-Right-Pointing-Pointer Without a sperm centriole, parthenogenetic oocytes produce centrioles and centrosomes. Black-Right-Pointing-Pointer Parthenogenetic human oocytes can develop and differentiate into mature cells. -- Abstract: In most animals, somatic cell centrosomes are inherited from the centriole of the fertilizing spermatozoa. The oocyte centriole degenerates during oogenesis, and completely disappears in metaphase II. Therefore, the embryos generated by in vitro parthenogenesis are supposed to develop without any centrioles. Exceptional acentriolar and/or acentrosomal developments are possible in mice and in some experimental cells; however, in most animals, the full developmental potential of parthenogenetic cells in vitro and the fate of their centrioles/centrosomes are not clearly understood. To predict the future of in vitro human parthenogenesis, we explored the centrioles/centrosomes in ovarian mature cystic teratoma cells by immunofluorescent staining and transmission electron microscopy. We confirmed the presence of centrioles and centrosomes in these well-known parthenogenetic ovarian tumor cells. Our findings clearly demonstrate that, even without a sperm centriole, parthenotes that develop from activated oocytes can produce their own centrioles/centrosomes, and can even develop into the well-differentiated mature tissue.

  16. Subcellular localization of (latent) transforming growth factor beta and the latent TGF-beta binding protein in rat hepatocytes and hepatic stellate cells.

    PubMed

    Roth-Eichhorn, S; Kühl, K; Gressner, A M

    1998-12-01

    Recently, the existence of the large latent transforming growth factor beta (TGF-beta) complex, consisting of TGF-beta, the N-terminal part of its precursor (latency-associated peptide [LAP]), and the latent TGF-beta binding protein (LTBP), was demonstrated in rat liver parenchymal cells (PC) and stellate cells (HSC). However, in contrast to HSC, in freshly isolated PC, no message of these proteins is detectable. This study was performed to investigate the subcellular distribution of the proteins forming the latent TGF-beta complex in PC and HSC from rat liver to obtain more information about their origin and potential intracellular functions. PC and HSC were isolated from rat liver by protease reperfusion and investigated for TGF-beta1,-2,-3, beta1-LAP, and LTBP-1 after cultivation using double-immunofluorescent staining, followed by high-resolution confocal microscopic analysis. Subcellular fractions obtained by standard differential centrifugation of rat liver homogenate were analyzed using a TGF-beta1 enzyme-linked immunosorbent assay (ELISA) and Western blotting for beta1-LAP and LTBP-1. By confocal microscopy, a diffuse distribution of TGF-beta and LAP in the cytoplasm of PC is noticed, whereas the LTBP immunostaining predominates at plasma membranes. In PC, distinct intracellular granules were superimposed with TGF-beta, LAP, and LTBP stainings identified as lysosomal compartments and mitochondria by ELISA and immunoblotting of subcellular fractions. In HSC, stainings of colocalized TGF-beta, LAP, and LTBP are strongest in the perinuclear area, indicating synthesis and secretion via endoplasmic reticulum and Golgi, respectively. Partially, the proteins were also found in HSC nuclei. During the transformation of HSC to myofibroblasts, LAP and LTBP become strongly colocalized with other components of the cytoskeletal network like smooth muscle--actin, desmin, and talin. The results confirm biochemical data about the existence and expression of the large latent

  17. Probing Meiotic Recombination and Aneuploidy of Single Sperm Cells by Whole Genome Sequencing

    PubMed Central

    Lu, Sijia; Zong, Chenghang; Fan, Wei; Yang, Mingyu; Li, Jinsen; Chapman, Alec R.; Zhu, Ping; Hu, Xuesong; Xu, Liya; Yan, Liying; Bai, Fan; Qiao, Jie; Tang, Fuchou; Li, Ruiqiang; Xie, X. Sunney

    2013-01-01

    Meiotic recombination creates genetic diversity and ensures segregation of homologous chromosomes. Previous population analyses yielded results averaged among individuals and impacted by evolutionary pressures. Here we sequenced 99 sperm from an Asian male using the newly developed amplification method—Multiple Annealing and Looping-Based Amplification Cycles (MALBAC)—to phase the personal genome and map at high resolution recombination events, which are non-uniformly distributed across the genome in the absence of selection pressure. The paucity of recombination near transcription start sites observed in individual sperm indicates such a phenomenon is intrinsic to the molecular mechanism of meiosis. Interestingly, a decreased crossover frequency in companion with an increase of autosomal aneuploidy is observable on a global per-sperm basis. PMID:23258895

  18. Combined thermotherapy and cryotherapy for efficient virus eradication: relation of virus distribution, subcellular changes, cell survival and viral RNA degradation in shoot tips.

    PubMed

    Wang, Qiaochun; Cuellar, Wilmer J; Rajamäki, Minna-Liisa; Hirata, Yukimasa; Valkonen, Jari P T

    2008-03-01

    Accumulation of viruses in vegetatively propagated plants causes heavy yield losses. Therefore, supply of virus-free planting materials is pivotal to sustainable crop production. In previous studies, Raspberry bushy dwarf virus (RBDV) was difficult to eradicate from raspberry (Rubus idaeus) using the conventional means of meristem tip culture. As shown in the present study, it was probably because this pollen-transmitted virus efficiently invades leaf primordia and all meristematic tissues except the least differentiated cells of the apical dome. Subjecting plants to thermotherapy prior to meristem tip culture heavily reduced viral RNA2, RNA3 and the coat protein in the shoot tips, but no virus-free plants were obtained. Therefore, a novel method including thermotherapy followed by cryotherapy was developed for efficient virus eradication. Heat treatment caused subcellular alterations such as enlargement of vacuoles in the more developed, virus-infected cells, which were largely eliminated following subsequent cryotherapy. Using this protocol, 20-36% of the treated shoot tips survived, 30-40% regenerated and up to 35% of the regenerated plants were virus-free, as tested by ELISA and reverse transcription loop-mediated isothermal amplification. Novel cellular and molecular insights into RBDV-host interactions and the factors influencing virus eradication were obtained, including invasion of shoot tips and meristematic tissues by RBDV, enhanced viral RNA degradation and increased sensitivity to freezing caused by thermotherapy, and subcellular changes and subsequent death of cells caused by cryotherapy. This novel procedure should be helpful with many virus-host combinations in which virus eradication by conventional means has proven difficult.

  19. Plasma effects on subcellular structures

    SciTech Connect

    Gweon, Bomi; Kim, Dan Bee; Jung, Heesoo; Choe, Wonho; Kim, Daeyeon; Shin, Jennifer H.

    2010-03-08

    Atmospheric pressure helium plasma treated human hepatocytes exhibit distinctive zones of necrotic and live cells separated by a void. We propose that plasma induced necrosis is attributed to plasma species such as oxygen radicals, charged particles, metastables and/or severe disruption of charged cytoskeletal proteins. Interestingly, uncharged cytoskeletal intermediate filaments are only minimally disturbed by plasma, elucidating the possibility of plasma induced electrostatic effects selectively destroying charged proteins. These bona fide plasma effects, which inflict alterations in specific subcellular structures leading to necrosis and cellular detachment, were not observed by application of helium flow or electric field alone.

  20. Sperm Motility in Flow

    NASA Astrophysics Data System (ADS)

    Guasto, Jeffrey; Juarez, Gabriel; Stocker, Roman

    2012-11-01

    A wide variety of plants and animals reproduce sexually by releasing motile sperm that seek out a conspecific egg, for example in the reproductive tract for mammals or in the water column for externally fertilizing organisms. Sperm are aided in their quest by chemical cues, but must also contend with hydrodynamic forces, resulting from laminar flows in reproductive tracts or turbulence in aquatic habitats. To understand how velocity gradients affect motility, we subjected swimming sperm to a range of highly-controlled straining flows using a cross-flow microfluidic device. The motion of the cell body and flagellum were captured through high-speed video microscopy. The effects of flow on swimming are twofold. For moderate velocity gradients, flow simply advects and reorients cells, quenching their ability to cross streamlines. For high velocity gradients, fluid stresses hinder the internal bending of the flagellum, directly inhibiting motility. The transition between the two regimes is governed by the Sperm number, which compares the external viscous stresses with the internal elastic stresses. Ultimately, unraveling the role of flow in sperm motility will lead to a better understanding of population dynamics among aquatic organisms and infertility problems in humans.

  1. Quantifying β-catenin subcellular dynamics and cyclin D1 mRNA transcription during Wnt signaling in single living cells

    PubMed Central

    Kafri, Pinhas; Hasenson, Sarah E; Kanter, Itamar; Sheinberger, Jonathan; Kinor, Noa; Yunger, Sharon; Shav-Tal, Yaron

    2016-01-01

    Signal propagation from the cell membrane to a promoter can induce gene expression. To examine signal transmission through sub-cellular compartments and its effect on transcription levels in individual cells within a population, we used the Wnt/β-catenin signaling pathway as a model system. Wnt signaling orchestrates a response through nuclear accumulation of β-catenin in the cell population. However, quantitative live-cell measurements in individual cells showed variability in nuclear β-catenin accumulation, which could occur in two waves, followed by slow clearance. Nuclear accumulation dynamics were initially rapid, cell cycle independent and differed substantially from LiCl stimulation, presumed to mimic Wnt signaling. β-catenin levels increased simultaneously at adherens junctions and the centrosome, and a membrane-centrosome transport system was revealed. Correlating β-catenin nuclear dynamics to cyclin D1 transcriptional activation showed that the nuclear accumulation rate of change of the signaling factor, and not actual protein levels, correlated with the transcriptional output of the pathway. DOI: http://dx.doi.org/10.7554/eLife.16748.001 PMID:27879202

  2. Current status and potential of morphometric sperm analysis

    PubMed Central

    Maroto-Morales, Alejandro; García-Álvarez, Olga; Ramón, Manuel; Martínez-Pastor, Felipe; Fernández-Santos, M Rocío; Soler, A Josefa; Garde, José Julián

    2016-01-01

    The spermatozoon is the most diverse cell type known and this diversity is considered to reflect differences in sperm function. How the diversity in sperm morphology arose during speciation and what role the different specializations play in sperm function, however, remain incompletely characterized. This work reviews the hypotheses proposed to explain sperm morphological evolution, with a focus on some aspects of sperm morphometric evaluation; the ability of morphometrics to predict sperm cryoresistance and male fertility is also discussed. For this, the evaluation of patterns of change of sperm head morphometry throughout a process, instead of the study of the morphometric characteristics of the sperm head at different stages, allows a better identification of the males with different sperm cryoconservation ability. These new approaches, together with more studies employing a greater number of individuals, are needed to obtain novel results concerning the role of sperm morphometry on sperm function. Future studies should aim at understanding the causes of sperm design diversity and the mechanisms that generate them, giving increased attention to other sperm structures besides the sperm head. The implementation of scientific and technological advances could benefit the simultaneous examination of sperm phenotype and sperm function, demonstrating that sperm morphometry could be a useful tool for sperm assessment. PMID:27678465

  3. MicroRNA-29a inhibited epididymal epithelial cell proliferation by targeting nuclear autoantigenic sperm protein (NASP).

    PubMed

    Ma, Wubin; Xie, Shengsong; Ni, Minjie; Huang, Xingxu; Hu, Shuanggang; Liu, Qiang; Liu, Aihua; Zhang, Jinsong; Zhang, Yonglian

    2012-03-23

    Cell proliferation often decreases gradually during postnatal development of some organs. However, the underlying molecular mechanisms remain unclear. Epididymis, playing important roles in sperm maturation, is a typical organ of this type, which displays a decreased proliferation during postnatal development and even ceased at the adult stage. Here, epididymis was employed as a model to explore the underlying mechanisms. We profiled the microRNA and mRNA expression of newborn (1 day) and adult (90 day) rat epididymis by microarray analysis, and found that the level of miR-29a was dramatically up-regulated during postnatal development of rat epididymis. Subsequent investigations demonstrated that overexpression of miR-29a inhibited the proliferation of epididymal epithelial cells in vitro. The nuclear autoantigenic sperm protein (NASP), a novel target of miR-29a, was significantly down-regulated during postnatal development of rat epididymis. Further analysis showed that silence of NASP mimicked the anti-proliferation effect of miR-29a, whereas overexpression of this protein attenuated the effect of miR-29a. As in rat epididymis, miR-29a was up-regulated and Nasp was down-regulated during postnatal development of mouse epididymis, heart, liver, and lung. Moreover, miR-29a can also inhibit the proliferation of cancer cells by targeting Nasp. Thus, an increase of miR-29a, and hence decrease of Nasp, may contribute to inhibit cell proliferation during postnatal organ development.

  4. Efficient isolation of sperm with high DNA integrity and stable chromatin packaging by a combination of density-gradient centrifugation and magnetic-activated cell sorting

    PubMed Central

    Kwak, Su-Jin; Kim, Seok-Gi; Kim, Youn-Young; Park, Ji-Young; Yoo, Chang-Seok; Park, Il-Hae; Sun, Hong-Gil; Kim, Jae-Won; Lee, Kyeong-Ho

    2016-01-01

    Objective This study was carried out to investigate the correlations of the sperm DNA fragmentation index (DFI) with semen parameters and apoptosis, and to investigate the effects of density-gradient centrifugation (DGC) and magnetic-activated cell sorting (MACS) on reducing the proportion of sperm with DNA fragmentation and protamine deficiency. Methods Semen analysis and a sperm DNA fragmentation assay were performed to assess the correlations between semen parameters and the DFI in 458 semen samples. Sperm with progressive motility or non-apoptosis were isolated by DGC or MACS, respectively, in 29 normozoospermic semen samples. The effects of DGC or MACS alone and of DGC and MACS combined on reducing the amount of sperm in the sample with DNA fragmentation and protamine deficiency were investigated. Results The sperm DFI showed a significant correlation (r=–0.347, p<0.001) with sperm motility and morphology (r=–0.114, p<0.05) but not with other semen parameters. The DFI (11.5%±2.0%) of semen samples was significantly reduced by DGC (8.1%±4.1%) or MACS alone (7.4%±3.9%) (p<0.05). The DFI was significantly further reduced by a combination of DGC and MACS (4.1%±1.3%, p<0.05). Moreover, the combination of DGC and MACS (1.6%±1.1%, p<0.05) significantly reduced the protamine deficiency rate of semen samples compared to DGC (4.4%±3.2%) or MACS alone (3.4%±2.2%). Conclusion The combination of DGC and MACS may be an effective method to isolate high-quality sperm with progressive motility, non-apoptosis, high DNA integrity, and low protamine deficiency in clinical use. PMID:28090458

  5. Tris-egg yolk-glycerol (TEY) extender developed for freezing dog semen is a good option to cryopreserve bovine epididymal sperm cells.

    PubMed

    Lopes, G; Soares, L; Ferreira, P; Rocha, A

    2015-02-01

    Cryopreservation of epididymal spermatozoa is often performed after shipping the excised testis-epididymis complexes, under refrigeration, to a specialized laboratory. However, epididymal spermatozoa can be collected immediately after excision of the epididymis and sent extended and refrigerated to a laboratory for cryopreservation. In this experiment, we evaluated the effect of both methods of cold storage bovine epididymal spermatozoa as well as of two different extenders on spermatozoa characteristics after freeze-thawing. For that, spermatozoa collected from the caudae epididymis of 19 bulls were extended and cryopreserved in either AndroMed(®) or a Tris-egg yolk (TEY)-based extender. Cryopreservation of sperm cells was performed immediately after castration (Group A, n = 9) or after cold storage for 24 h diluted in the two extenders and (Group B, n = 9) and also after cold storage for 24 h within the whole epididymis (Group C, n = 10). Sperm subjective progressive motility (light microscopy), plasma membrane integrity (hypoosmotic swelling test) and sperm viability (eosin-nigrosin) were evaluated. In vitro fertilization and culture (IVF) was performed to assess the blastocyst rate. No differences (p > 0.05) were observed on post-thaw sperm parameters between samples from Group A, B and C. TEY extended samples presented a higher (p < 0.01) percentage of progressive motile and live sperm, than those extended in AndroMed(®) . Blastocyst rate after IVF differed only (p < 0.05) between the reference group (IVF performed with frozen semen with known in vitro fertility) and Group A extended in AndroMed(®) . We conclude that when cryopreservation facilities are distant from the collection site, bovine epididymal sperm can be shipped chilled overnight either within the epididymal tail or after dilution without deleterious effect on post-thaw sperm quality. TEY extender was more suitable for cold storage and freezing bovine epididymal sperm, than the commercial

  6. Characteristics of stallion epididymal spermatozoa at collection and effect of two refrigeration protocols on the quality of the frozen/thawed sperm cells.

    PubMed

    Guimarães, T; Lopes, G; Ferreira, P; Leal, I; Rocha, A

    2012-12-01

    Cryopreservation of epididymal spermatozoa is a useful tool to preserve genetic material of valuable stallions after emergency castration or unexpected death. For that, testicles and epididymides are generally sent refrigerated to the laboratory. Collection of epididymal spermatozoa is a simple procedure that reduces the volume of the material to be shipped, and may improve the quality of the chilled epididymal sperm cells. In the present study we compared the characteristics of frozen/thawed epididymal spermatozoa after refrigeration of the epididymis or after direct refrigeration of the extended epididymal sperm cells. Ejaculated sperm samples were obtained from 10 healthy stallions with at least 15 days of sexual rest, before routine orchiectomies. Spermatozoa were recovered from the epididymal tail immediately after castration (EPI), after refrigeration of the epididymis for 24h at 4°C (EPI R) and recovered from epididymal tail immediately after castration and stored for 24h at 4°C (EPI RR). Total motility, straight-line velocity, percentage of rapid cells, viability and morphological defects were similar (p>0.05) among different treatments, and post-thaw viability was higher (p<0.05) in EPI than in the ejaculated sperm. The similarity of post-thaw parameters led us to conclude that immediate collection and refrigeration of the epididymal sperm cells or refrigeration of the whole epididymis are equally efficient as a means of transporting material for 24h before cryopreservation of epididymal spermatozoa.

  7. Investigation of the effects of cell model and subcellular location of gold nanoparticles on nuclear dose enhancement factors using Monte Carlo simulation

    SciTech Connect

    Cai, Zhongli; Chattopadhyay, Niladri; Kwon, Yongkyu Luke; Pignol, Jean-Philippe; Lechtman, Eli; Reilly, Raymond M.

    2013-11-15

    Purpose: The authors’ aims were to model how various factors influence radiation dose enhancement by gold nanoparticles (AuNPs) and to propose a new modeling approach to the dose enhancement factor (DEF).Methods: The authors used Monte Carlo N-particle (MCNP 5) computer code to simulate photon and electron transport in cells. The authors modeled human breast cancer cells as a single cell, a monolayer, or a cluster of cells. Different numbers of 5, 30, or 50 nm AuNPs were placed in the extracellular space, on the cell surface, in the cytoplasm, or in the nucleus. Photon sources examined in the simulation included nine monoenergetic x-rays (10–100 keV), an x-ray beam (100 kVp), and {sup 125}I and {sup 103}Pd brachytherapy seeds. Both nuclear and cellular dose enhancement factors (NDEFs, CDEFs) were calculated. The ability of these metrics to predict the experimental DEF based on the clonogenic survival of MDA-MB-361 human breast cancer cells exposed to AuNPs and x-rays were compared.Results: NDEFs show a strong dependence on photon energies with peaks at 15, 30/40, and 90 keV. Cell model and subcellular location of AuNPs influence the peak position and value of NDEF. NDEFs decrease in the order of AuNPs in the nucleus, cytoplasm, cell membrane, and extracellular space. NDEFs also decrease in the order of AuNPs in a cell cluster, monolayer, and single cell if the photon energy is larger than 20 keV. NDEFs depend linearly on the number of AuNPs per cell. Similar trends were observed for CDEFs. NDEFs using the monolayer cell model were more predictive than either single cell or cluster cell models of the DEFs experimentally derived from the clonogenic survival of cells cultured as a monolayer. The amount of AuNPs required to double the prescribed dose in terms of mg Au/g tissue decreases as the size of AuNPs increases, especially when AuNPs are in the nucleus and the cytoplasm. For 40 keV x-rays and a cluster of cells, to double the prescribed x-ray dose (NDEF = 2

  8. Redox regulation of mammalian sperm capacitation

    PubMed Central

    O’Flaherty, Cristian

    2015-01-01

    Capacitation is a series of morphological and metabolic changes necessary for the spermatozoon to achieve fertilizing ability. One of the earlier happenings during mammalian sperm capacitation is the production of reactive oxygen species (ROS) that will trigger and regulate a series of events including protein phosphorylation, in a time-dependent fashion. The identity of the sperm oxidase responsible for the production of ROS involved in capacitation is still elusive, and several candidates are discussed in this review. Interestingly, ROS-induced ROS formation has been described during human sperm capacitation. Redox signaling during capacitation is associated with changes in thiol groups of proteins located on the plasma membrane and subcellular compartments of the spermatozoon. Both, oxidation of thiols forming disulfide bridges and the increase on thiol content are necessary to regulate different sperm proteins associated with capacitation. Reducing equivalents such as NADH and NADPH are necessary to support capacitation in many species including humans. Lactate dehydrogenase, glucose-6-phospohate dehydrogenase, and isocitrate dehydrogenase are responsible in supplying NAD (P) H for sperm capacitation. Peroxiredoxins (PRDXs) are newly described enzymes with antioxidant properties that can protect mammalian spermatozoa; however, they are also candidates for assuring the regulation of redox signaling required for sperm capacitation. The dysregulation of PRDXs and of enzymes needed for their reactivation such as thioredoxin/thioredoxin reductase system and glutathione-S-transferases impairs sperm motility, capacitation, and promotes DNA damage in spermatozoa leading to male infertility. PMID:25926608

  9. Subcellular localization of caspase-3 activation correlates with changes in apoptotic morphology in MOLT-4 leukemia cells exposed to X-ray irradiation.

    PubMed

    Feng, Yongdong; Hu, Junbo; Xie, Daxin; Qin, Jichao; Zhong, Yisheng; Li, Xiaolan; Xiao, Wei; Wu, Jianhong; Tao, Deding; Zhang, Manchao; Zhu, Yunfeng; Song, Yuping; Reed, Eddie; Li, Qingdi Q; Gong, Jianping

    2005-09-01

    Caspase-3 is a critical effector caspase for apoptosis, which cleaves proteins, including cytoskeletal and associated proteins, kinases, and members of the Bcl-2 family of apoptosis-related proteins. This leads to changes in apoptotic morphology, such as membrane externalization and cytoplasm and nuclear condensation. It has been reported that pro-caspase-3 is activated in the cytosol. However, it remains obscure how caspase-3 activation correlates to serial changes in cell morphology during apoptosis. The current study was therefore undertaken to assess the relationship between caspase-3 activation and its subcellular localization and alterations in apoptotic morphology in MOLT-4 human leukemia cells exposed to X-ray irradiation. Fluorescence labeled inhibitor of caspases (FLICA) was used to detect caspase-3 activity in apoptotic cells in this project; cell morphology and caspase-3 sub-localization were determined by confocal microscopy. Our data showed that MOLT-4 cells presented typical morphological changes in apoptosis, such as membrane reversion, DNA fragmentation, and formation of apoptotic cell bodies following 10 Gray (Gy) of X-ray irradiation. Caspase-3 was activated 2 h after X-ray irradiation, and its activity increased markedly after 4-6-h exposure. Membrane reversion in MOLT-4 leukemia cells was detected by Annexin V assay at 4 h following X-ray irradiation, 2 h after the elevated caspase-3 activity was measured. Cytologically, activation of caspase-3 was first observed close to the inside surface of the cellular membrane, then transferred to the cytoplasm, and finally translocated to the nuclear region. We conclude that caspase-3 is activated in MOLT-4 cells following exposure to X-rays, and that the enhanced caspase-3 activity and its sub-localization shifting is correlated to changes in apoptotic morphology. The spatial shift of activated caspase-3 in X-ray-induced apoptotic MOLT-4 leukemia cells is a process of crucial importance for apoptosis.

  10. Virtual azoospermia and cryptozoospermia--fresh/frozen testicular or ejaculate sperm for better IVF outcome?

    PubMed

    Hauser, Ron; Bibi, Guy; Yogev, Leah; Carmon, Ariella; Azem, Foad; Botchan, Amnon; Yavetz, Haim; Klieman, Sandra E; Lehavi, Ofer; Amit, Ami; Ben-Yosef, Dalit

    2011-01-01

    Men diagnosed as having azoospermia occasionally have a few mature sperm cells in other ejaculates. Other men may have constant, yet very low quality and quantity of sperm cells in their ejaculates, resulting in poor intracytoplasmic sperm injection (ICSI) outcome. It has not been conclusively established which source of sperm cells is preferable for ICSI when both ejaculate and testicular (fresh or frozen) sperm cells are available. It is also unclear whether there is any advantage of fresh over frozen sperm if testicular sperm is to be used. We used ejaculate, testicular (fresh or frozen) sperm cells, or both for ICSI in 13 couples. Five of these couples initially underwent ICSI by testicular sperm extraction, because the males had total azoospermia, and in later cycles with ejaculate sperm cells. Ejaculate sperm cells were initially used for ICSI in the other 8 patients, and later with testicular sperm cells. The fertilization rate was significantly higher when fresh or frozen-thawed testicular sperm cells were used than when ejaculated sperm cells were used. Likewise, the quality of the embryos from testicular (fresh and frozen) sperm was higher than from ejaculated sperm (65.3% vs 53.2%, respectively, P < .05). The use of fresh testicular sperm yielded better implantation rates than both frozen testicular sperm and ejaculate. Therefore, fresh testicular sperm should be considered first for ICSI in patients with virtual azoospermia or cryptozoospermia because of their superior fertility.

  11. Recent advances in imaging subcellular processes.

    PubMed

    Myers, Kenneth A; Janetopoulos, Christopher

    2016-01-01

    Cell biology came about with the ability to first visualize cells. As microscopy techniques advanced, the early microscopists became the first cell biologists to observe the inner workings and subcellular structures that control life. This ability to see organelles within a cell provided scientists with the first understanding of how cells function. The visualization of the dynamic architecture of subcellular structures now often drives questions as researchers seek to understand the intricacies of the cell. With the advent of fluorescent labeling techniques, better and new optical techniques, and more sensitive and faster cameras, a whole array of questions can now be asked. There has been an explosion of new light microscopic techniques, and the race is on to build better and more powerful imaging systems so that we can further our understanding of the spatial and temporal mechanisms controlling molecular cell biology.

  12. Recent advances in imaging subcellular processes

    PubMed Central

    Myers, Kenneth A.; Janetopoulos, Christopher

    2016-01-01

    Cell biology came about with the ability to first visualize cells. As microscopy techniques advanced, the early microscopists became the first cell biologists to observe the inner workings and subcellular structures that control life. This ability to see organelles within a cell provided scientists with the first understanding of how cells function. The visualization of the dynamic architecture of subcellular structures now often drives questions as researchers seek to understand the intricacies of the cell. With the advent of fluorescent labeling techniques, better and new optical techniques, and more sensitive and faster cameras, a whole array of questions can now be asked. There has been an explosion of new light microscopic techniques, and the race is on to build better and more powerful imaging systems so that we can further our understanding of the spatial and temporal mechanisms controlling molecular cell biology. PMID:27408708

  13. Intracellular translocation and differential accumulation of cell-penetrating peptides in bovine spermatozoa: evaluation of efficient delivery vectors that do not compromise human sperm motility

    PubMed Central

    Jones, Sarah; Lukanowska, Monika; Suhorutsenko, Julia; Oxenham, Senga; Barratt, Christopher; Publicover, Steven; Copolovici, Dana Maria; Langel, Ülo; Howl, John

    2013-01-01

    STUDY QUESTION Do cell penetrating peptides (CPPs) translocate into spermatozoa and, if so, could they be utilized to deliver a much larger protein cargo? SUMMARY ANSWER Chemically diverse polycationic CPPs rapidly and efficiently translocate into spermatozoa. They exhibit differential accumulation within intracellular compartments without detrimental influences upon cellular viability or motility but they are relatively ineffective in transporting larger proteins. WHAT IS ALREADY KNOWN Endocytosis, the prevalent route of protein internalization into eukaryotic cells, is severely compromised in mature spermatozoa. Thus, the translocation of many bioactive agents into sperm is relatively inefficient. However, the delivery of bioactive moieties into mature spermatozoa could be significantly improved by the identification and utility of an efficient and inert vectorial delivery technology. STUDY DESIGN CPP translocation efficacies, their subsequent differential intracellular distribution and the influence of peptides upon viability were determined in bovine spermatozoa. Temporal analyses of sperm motility in the presence of exogenously CPPs utilized normozoospermic human donor samples. MATERIALS AND METHODS CPPs were prepared by manual, automated and microwave-enhanced solid phase synthesis. Confocal fluorescence microscopy determined the intracellular distribution of rhodamine-conjugated CPPs in spermatozoa. Quantitative uptake and kinetic analyses compared the translocation efficacies of chemically diverse CPPs and conjugates of biotinylated CPPs and avidin. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) conversion assays were employed to analyse the influence of CPPs upon sperm cell viability and sperm class assays determined the impact of CPPs on motility in capacitated and non-capacitated human samples. MAIN RESULTS Chemically heterogeneous CPPs readily translocated into sperm to accumulate within

  14. Human sperm rheotaxis: a passive physical process

    PubMed Central

    Zhang, Zhuoran; Liu, Jun; Meriano, Jim; Ru, Changhai; Xie, Shaorong; Luo, Jun; Sun, Yu

    2016-01-01

    A long-standing question in natural reproduction is how mammalian sperm navigate inside female reproductive tract and finally reach the egg cell, or oocyte. Recently, fluid flow was proposed as a long–range guidance cue for sperm navigation. Coitus induces fluid flow from oviduct to uterus, and sperm align themselves against the flow direction and swim upstream, a phenomenon termed rheotaxis. Whether sperm rheotaxis is a passive process dominated by fluid mechanics, or sperm actively sense and adapt to fluid flow remains controversial. Here we report the first quantitative study of sperm flagellar motion during human sperm rheotaxis and provide direct evidence indicating that sperm rheotaxis is a passive process. Experimental results show that there is no significant difference in flagellar beating amplitude and asymmetry between rheotaxis-turning sperm and those sperm swimming freely in the absence of fluid flow. Additionally, fluorescence image tracking shows no Ca2+ influx during sperm rheotaxis turning, further suggesting there is no active signal transduction during human sperm rheotaxis. PMID:27005727

  15. Human sperm rheotaxis: a passive physical process

    NASA Astrophysics Data System (ADS)

    Zhang, Zhuoran; Liu, Jun; Meriano, Jim; Ru, Changhai; Xie, Shaorong; Luo, Jun; Sun, Yu

    2016-03-01

    A long-standing question in natural reproduction is how mammalian sperm navigate inside female reproductive tract and finally reach the egg cell, or oocyte. Recently, fluid flow was proposed as a long–range guidance cue for sperm navigation. Coitus induces fluid flow from oviduct to uterus, and sperm align themselves against the flow direction and swim upstream, a phenomenon termed rheotaxis. Whether sperm rheotaxis is a passive process dominated by fluid mechanics, or sperm actively sense and adapt to fluid flow remains controversial. Here we report the first quantitative study of sperm flagellar motion during human sperm rheotaxis and provide direct evidence indicating that sperm rheotaxis is a passive process. Experimental results show that there is no significant difference in flagellar beating amplitude and asymmetry between rheotaxis-turning sperm and those sperm swimming freely in the absence of fluid flow. Additionally, fluorescence image tracking shows no Ca2+ influx during sperm rheotaxis turning, further suggesting there is no active signal transduction during human sperm rheotaxis.

  16. Why small males have big sperm: dimorphic squid sperm linked to alternative mating behaviours

    PubMed Central

    2011-01-01

    Background Sperm cells are the target of strong sexual selection that may drive changes in sperm structure and function to maximize fertilisation success. Sperm evolution is regarded to be one of the major consequences of sperm competition in polyandrous species, however it can also be driven by adaptation to the environmental conditions at the site of fertilization. Strong stabilizing selection limits intra-specific variation, and therefore polymorphism, among fertile sperm (eusperm). Here we analyzed reproductive morphology differences among males employing characteristic alternative mating behaviours, and so potentially different conditions of sperm competition and fertilization environment, in the squid Loligo bleekeri. Results Large consort males transfer smaller (average total length = 73 μm) sperm to a female's internal sperm storage location, inside the oviduct; whereas small sneaker males transfer larger (99 μm) sperm to an external location around the seminal receptacle near the mouth. No significant difference in swimming speed was observed between consort and sneaker sperm. Furthermore, sperm precedence in the seminal receptacle was not biased toward longer sperm, suggesting no evidence for large sperm being favoured in competition for space in the sperm storage organ among sneaker males. Conclusions Here we report the first case, in the squid Loligo bleekeri, where distinctly dimorphic eusperm are produced by different sized males that employ alternative mating behaviours. Our results found no evidence that the distinct sperm dimorphism was driven by between- and within-tactic sperm competition. We propose that presence of alternative fertilization environments with distinct characteristics (i.e. internal or external), whether or not in combination with the effects of sperm competition, can drive the disruptive evolution of sperm size. PMID:21831296

  17. Subcellular localization of the human papillomavirus 16 E7 oncoprotein in CaSki cells and its detection in cervical adenocarcinoma and adenocarcinoma in situ.

    PubMed

    Dreier, Kerstin; Scheiden, René; Lener, Barbara; Ehehalt, Daniela; Pircher, Haymo; Müller-Holzner, Elisabeth; Rostek, Ursula; Kaiser, Andreas; Fiedler, Marc; Ressler, Sigrun; Lechner, Stefan; Widschwendter, Andreas; Even, Jos; Capesius, Catherine; Jansen-Dürr, Pidder; Zwerschke, Werner

    2011-01-05

    E7 is the major oncoprotein of high-risk human papillomaviruses (HPV) which causes cervical cancer. To date E7 oncoproteins have not been investigated in cervical adenocarcinoma. In this study we generated a rabbit monoclonal anti-HPV-16 E7 antibody, RabMab42-3, which recognizes a conformational epitope in the E7 carboxy-terminal zinc-finger resulting in a strong increase in the sensitivity for the detection of cell-associated HPV-16 E7 protein relative to conventional polyclonal anti-HPV-16 E7 antibodies. Using RabMab42-3, we show that the subcellular localization of endogenous HPV-16 E7 oncoprotein varies during the cell cycle in cervical cancer cells. Moreover, we demonstrate for the first time that the HPV-16 E7 oncoprotein is abundantly expressed in cervical adenocarcinoma in situ and adenocarcinoma, suggesting an important role of HPV-16 E7 for the development of these tumors. Our findings suggest that the HPV-16 E7 oncoprotein could be a useful marker for the detection of cervical adenocarcinoma and their precursors.

  18. cDNA cloning and characterization of Npap60: a novel rat nuclear pore-associated protein with an unusual subcellular localization during male germ cell differentiation.

    PubMed

    Fan, F; Liu, C P; Korobova, O; Heyting, C; Offenberg, H H; Trump, G; Arnheim, N

    1997-03-15

    We have cloned and characterized a cDNA, Npap60, encoding a rat nuclear pore-associated protein. The 3-kb cDNA was obtained by antibody screening of a rat testis expression library. The predicted NPAP60 contains 381 amino acids with a composition of 25.6% charged residues and is highly hydrophilic. The Npap60 gene appears to be conserved in mouse, rat, and human. Immunofluorescence studies with anti-NPAP60 fusion protein antibody show that the NPAP60 protein colocalizes with nuclear pore complexes in RAT1A cells. The expression of Npap60 is about 10-20 times higher in rat testis than in somatic tissues. The subcellular localization of NPAP60 protein changes dramatically during male germ cell differentiation, from nuclear pore complex-like staining in spermatocytes to whole nucleus staining in spermatids and finally to a nuclear surface staining in mature spermatozoa. These changes are temporally and spatially related to nuclear reorganization during male germ cell differentiation.

  19. Scaling factors: transcription factors regulating subcellular domains.

    PubMed

    Mills, Jason C; Taghert, Paul H

    2012-01-01

    Developing cells acquire mature fates in part by selective (i.e. qualitatively different) expression of a few cell-specific genes. However, all cells share the same basic repertoire of molecular and subcellular building blocks. Therefore, cells must also specialize according to quantitative differences in cell-specific distributions of those common molecular resources. Here we propose the novel hypothesis that evolutionarily-conserved transcription factors called scaling factors (SFs) regulate quantitative differences among mature cell types. SFs: (1) are induced during late stages of cell maturation; (2) are dedicated to specific subcellular domains; and, thus, (3) allow cells to emphasize specific subcellular features. We identify candidate SFs and discuss one in detail: MIST1 (BHLHA15, vertebrates)/DIMM (CG8667, Drosophila); professional secretory cells use this SF to scale up regulated secretion. Because cells use SFs to develop their mature properties and also to adapt them to ever-changing environmental conditions, SF aberrations likely contribute to diseases of adult onset.

  20. Sperm Competition, Sperm Numbers and Sperm Quality in Muroid Rodents

    PubMed Central

    Gómez Montoto, Laura; Magaña, Concepción; Tourmente, Maximiliano; Martín-Coello, Juan; Crespo, Cristina; Luque-Larena, Juan José

    2011-01-01

    Sperm competition favors increases in relative testes mass and production efficiency, and changes in sperm phenotype that result in faster swimming speeds. However, little is known about its effects on traits that contribute to determine the quality of a whole ejaculate (i.e., proportion of motile, viable, morphologically normal and acrosome intact sperm) and that are key determinants of fertilization success. Two competing hypotheses lead to alternative predictions: (a) sperm quantity and quality traits co-evolve under sperm competition because they play complementary roles in determining ejaculate's competitive ability, or (b) energetic constraints force trade-offs between traits depending on their relevance in providing a competitive advantage. We examined relationships between sperm competition levels, sperm quantity, and traits that determine ejaculate quality, in a comparative study of 18 rodent species using phylogenetically controlled analyses. Total sperm numbers were positively correlated to proportions of normal sperm, acrosome integrity and motile sperm; the latter three were also significantly related among themselves, suggesting no trade-offs between traits. In addition, testes mass corrected for body mass (i.e., relative testes mass), showed a strong association with sperm numbers, and positive significant associations with all sperm traits that determine ejaculate quality with the exception of live sperm. An “overall sperm quality” parameter obtained by principal component analysis (which explained 85% of the variance) was more strongly associated with relative testes mass than any individual quality trait. Overall sperm quality was as strongly associated with relative testes mass as sperm numbers. Thus, sperm quality traits improve under sperm competition in an integrated manner suggesting that a combination of all traits is what makes ejaculates more competitive. In evolutionary terms this implies that a complex network of genetic and

  1. PKM2 Subcellular Localization Is Involved in Oxaliplatin Resistance Acquisition in HT29 Human Colorectal Cancer Cell Lines

    PubMed Central

    Ginés, Alba; Bystrup, Sara; Ruiz de Porras, Vicenç; Guardia, Cristina; Musulén, Eva; Martínez-Cardús, Anna; Manzano, José Luis; Layos, Laura; Abad, Albert; Martínez-Balibrea, Eva

    2015-01-01

    Chemoresistance is the main cause of treatment failure in advanced colorectal cancer (CRC). However, molecular mechanisms underlying this phenomenon remain to be elucidated. In a previous work we identified low levels of PKM2 as a putative oxaliplatin-resistance marker in HT29 CRC cell lines and also in patients. In order to assess how PKM2 influences oxaliplatin response in CRC cells, we silenced PKM2 using specific siRNAs in HT29, SW480 and HCT116 cells. MTT test demonstrated that PKM2 silencing induced resistance in HT29 and SW480 cells and sensitivity in HCT116 cells. Same experiments in isogenic HCT116 p53 null cells and double silencing of p53 and PKM2 in HT29 cells failed to show an influence of p53. By using trypan blue stain and FITC-Annexin V/PI tests we detected that PKM2 knockdown was associated with an increase in cell viability but not with a decrease in apoptosis activation in HT29 cells. Fluorescence microscopy revealed PKM2 nuclear translocation in response to oxaliplatin in HCT116 and HT29 cells but not in OXA-resistant HTOXAR3 cells. Finally, by using a qPCR Array we demonstrated that oxaliplatin and PKM2 silencing altered cell death gene expression patterns including those of BMF, which was significantly increased in HT29 cells in response to oxaliplatin, in a dose and time-dependent manner, but not in siPKM2-HT29 and HTOXAR3 cells. BMF gene silencing in HT29 cells lead to a decrease in oxaliplatin-induced cell death. In conclusion, our data report new non-glycolytic roles of PKM2 in response to genotoxic damage and proposes BMF as a possible target gene of PKM2 to be involved in oxaliplatin response and resistance in CRC cells. PMID:25955657

  2. Daily exposure to summer temperatures affects the motile subpopulation structure of epididymal sperm cells but not male fertility in an in vivo rabbit model.

    PubMed

    Maya-Soriano, M J; Taberner, E; Sabés-Alsina, M; Ramon, J; Rafel, O; Tusell, L; Piles, M; López-Béjar, M

    2015-08-01

    High temperatures have negative effects on sperm quality leading to temporary or permanent sterility. The aim of the study was to assess the effect of long exposure to summer circadian heat stress cycles on sperm parameters and the motile subpopulation structure of epididymal sperm cells from rabbit bucks. Twelve White New Zealand rabbit bucks were exposed to a daily constant temperature of the thermoneutral zone (from 18 °C to 22 °C; control group) or exposed to a summer circadian heat stress cycles (30 °C, 3 h/day; heat stress group). Spermatozoa were flushed from the epididymis and assessed for sperm quality parameters at recovery. Sperm total motility and progressivity were negatively affected by high temperatures (P < 0.05), as were also specific motility parameters (curvilinear velocity, linear velocity, mean velocity, straightness coefficient, linearity coefficient, wobble coefficient, and frequency of head displacement; P < 0.05, but not the mean amplitude of lateral head displacement). Heat stress significantly increased the percentage of less-motile sperm subpopulations, although the percentage of the high-motile subpopulation was maintained, which is consistent with the fact that no effect was detected on fertility rates. However, prolificacy was reduced in females submitted to heat stress when inseminated by control bucks. In conclusion, our results suggest that environmental high temperatures are linked to changes in the proportion of motile sperm subpopulations of the epididymis, although fertility is still preserved despite the detrimental effects of heat stress. On the other hand, prolificacy seems to be affected by the negative effects of high temperatures, especially by altering female reproduction.

  3. Evaluation of sperm tests as indicators of germ-cell damage in men exposed to chemical or physical agents

    SciTech Connect

    Wyrobek, A.J.; Watchmaker, G.; Gordon, L.

    1983-06-15

    As reviewed here, at least 89 chemical exposures have been studied for their effects on human spermatogenesis using sperm tests, with the majority showing some effect on sperm count, motility, or morphology. Approximately 85% of these exposures were to experimental or therapeutic drugs, 10% to occupational or environmental agents, and 5% to recreational drugs. This paper briefly describes the more common sperm-based methods and reviews some of their applications. It also includes guidelines for undertaking a human sperm study, as well as a discussion of the predictive value of induced sperm changes, an evaluation of the role of animal sperm tests, and a summary of the advantages and disadvantages of the sperm tests.

  4. Two birds, one stone: dual targeting of the cancer cell surface and subcellular mitochondria by the galectin-3-binding peptide G3-C12.

    PubMed

    Sun, Wei; Li, Lian; Yang, Qing-Qing; Zhang, Zhi-Rong; Huang, Yuan

    2017-01-09

    Active tumor-targeting approaches using specific ligands have drawn considerable attention over the years. However, a single ligand often fails to simultaneously target the cancer cell surface and subcellular organelles, which limits the maximum therapeutic efficacy of delivered drugs. We describe a polymeric delivery system modified with the G3-C12 peptide for sequential dual targeting. In this study, galectin-3-targeted G3-C12 peptide was conjugated onto the N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer for the delivery of D(KLAKLAK)2 (KLA) peptide. G3-C12-HPMA-KLA exhibited increased receptor-mediated internalization into galectin-3-overexpressing PC-3 cells. Furthermore, G3-C12 peptide also directed HPMA-KLA conjugates to mitochondria. This occurred because the apoptosis signal triggered the accumulation of galectin-3 in mitochondria, and the G3-C12 peptide that specifically bound to galectin-3 was trafficked along with its receptor intracellularly. As a result, G3-C12-HPMA-KLA disrupted the mitochondrial membrane, increased the generation of reactive oxygen species (ROS) and induced cytochrome c release, which ultimately resulted in enhanced cytotoxicity. An in vivo study revealed that the G3-C12 peptide significantly enhanced the tumor accumulation of the KLA conjugate. In addition, G3-C12-HPMA-KLA exhibited the best therapeutic efficacy and greatly improved the animal survival rate. Our work demonstrates that G3-C12 is a promising ligand with dual-targeting functionality.

  5. A multi-channel device for high-density target-selective stimulation and long-term monitoring of cells and subcellular features in C. elegans.

    PubMed

    Lee, Hyewon; Kim, Shin Ae; Coakley, Sean; Mugno, Paula; Hammarlund, Marc; Hilliard, Massimo A; Lu, Hang

    2014-12-07

    Selective cell ablation can be used to identify neuronal functions in multicellular model organisms such as Caenorhabditis elegans. The optogenetic tool KillerRed facilitates selective ablation by enabling light-activated damage of cell or subcellular components in a temporally and spatially precise manner. However, the use of KillerRed requires stimulating (5 min-1 h), culturing (~24 h) and imaging (often repeatedly) a large number of individual animals. Current manual manipulation methods are limited by their time-consuming, labor-intensive nature, and their usage of anesthetics. To facilitate large-scale selective ablation, culturing, and repetitive imaging, we developed a densely-packed multi-channel device and used it to perform high-throughput neuronal ablation on KillerRed-expressing animals. The ability to load worms in identical locations with high loading efficiency allows us to ablate selected neurons in multiple worms simultaneously. Our device also enables continuous observation of animals for 24 h following KillerRed activation, and allows the animals to be recovered for behavioural assays. We expect this multi-channel device to facilitate a broad range of long-term imaging and selective illumination experiments in neuroscience.

  6. In vivo subcellular localization of Mal de Río Cuarto virus (MRCV) non-structural proteins in insect cells reveals their putative functions.

    PubMed

    Maroniche, Guillermo A; Mongelli, Vanesa C; Llauger, Gabriela; Alfonso, Victoria; Taboga, Oscar; del Vas, Mariana

    2012-09-01

    The in vivo subcellular localization of Mal de Río Cuarto virus (MRCV, Fijivirus, Reoviridae) non-structural proteins fused to GFP was analyzed by confocal microscopy. P5-1 showed a cytoplasmic vesicular-like distribution that was lost upon deleting its PDZ binding TKF motif, suggesting that P5-1 interacts with cellular PDZ proteins. P5-2 located at the nucleus and its nuclear import was affected by the deletion of its basic C-termini. P7-1 and P7-2 also entered the nucleus and therefore, along with P5-2, could function as regulators of host gene expression. P6 located in the cytoplasm and in perinuclear cloud-like inclusions, was driven to P9-1 viroplasm-like structures and co-localized with P7-2, P10 and α-tubulin, suggesting its involvement in viroplasm formation and viral intracellular movement. Finally, P9-2 was N-glycosylated and located at the plasma membrane in association with filopodia-like protrusions containing actin, suggesting a possible role in virus cell-to-cell movement and spread.

  7. In vivo subcellular localization of Mal de Rio Cuarto virus (MRCV) non-structural proteins in insect cells reveals their putative functions

    SciTech Connect

    Maroniche, Guillermo A.; Mongelli, Vanesa C.; Llauger, Gabriela; Alfonso, Victoria; Taboga, Oscar

    2012-09-01

    The in vivo subcellular localization of Mal de Rio Cuarto virus (MRCV, Fijivirus, Reoviridae) non-structural proteins fused to GFP was analyzed by confocal microscopy. P5-1 showed a cytoplasmic vesicular-like distribution that was lost upon deleting its PDZ binding TKF motif, suggesting that P5-1 interacts with cellular PDZ proteins. P5-2 located at the nucleus and its nuclear import was affected by the deletion of its basic C-termini. P7-1 and P7-2 also entered the nucleus and therefore, along with P5-2, could function as regulators of host gene expression. P6 located in the cytoplasm and in perinuclear cloud-like inclusions, was driven to P9-1 viroplasm-like structures and co-localized with P7-2, P10 and {alpha}-tubulin, suggesting its involvement in viroplasm formation and viral intracellular movement. Finally, P9-2 was N-glycosylated and located at the plasma membrane in association with filopodia-like protrusions containing actin, suggesting a possible role in virus cell-to-cell movement and spread.

  8. Subcellular sites of processing of precursors to neurosecretory peptides in the bag cells of Aplysia: inferences from the effects of monensin, FCCP, and chloroquine.

    PubMed

    Yates, M E; Berry, R W

    1984-03-01

    The effects of the sodium ionophore monensin were examined in the bag cells of Aplysia californica in order to identify the subcellular sites of processing of precursors to their neurosecretory products. Incubation of bag cells in media containing 10 microM monensin led to a marked disruption of the morphology of the Golgi apparatus without affecting that of other organelles. Exposure of bag cells to monensin led to a significant impairment of processing of the largest precursor and of an intermediate protein which gives rise to the immediate precursors to the final secreted products, the egg-laying hormone (ELH) and the acidic peptide (AP). Furthermore, ELH and AP were never produced in the presence of monensin during the time course of these experiments. When axonal transport was allowed to proceed, the contents of bag-cell terminals indicated that the intermediate protein is the first to be packaged in Golgi-derived vesicles, and in monensin-treated cells may be transported without being processed further. In contrast to these results, the protonophore FCCP-impaired precursor and intermediate cleavage equally, indicating that monensin and FCCP have different effects on intracellular transport and precursor processing. These data are interpreted to indicate that the largest ELH-AP precursor is normally processed within the Golgi apparatus, and that the disruption of this organelle induced by monensin produces the impairment seen in its processing. The impairment of cleavage of the intermediate species, and the blockade of production of AP and ELH, are probably the result of monensin-induced impairment of production of proteolytically competent secretory granules by the Golgi apparatus.

  9. Subcellular analysis by laser ablation electrospray ionization mass spectrometry

    DOEpatents

    Vertes, Akos; Stolee, Jessica A; Shrestha, Bindesh

    2014-12-02

    In various embodiments, a method of laser ablation electrospray ionization mass spectrometry (LAESI-MS) may generally comprise micro-dissecting a cell comprising at least one of a cell wall and a cell membrane to expose at least one subcellular component therein, ablating the at least one subcellular component by an infrared laser pulse to form an ablation plume, intercepting the ablation plume by an electrospray plume to form ions, and detecting the ions by mass spectrometry.

  10. Understanding the Remodelling of Cell Walls during Brachypodium distachyon Grain Development through a Sub-Cellular Quantitative Proteomic Approach

    PubMed Central

    Francin-Allami, Mathilde; Lollier, Virginie; Pavlovic, Marija; San Clemente, Hélène; Rogniaux, Hélène; Jamet, Elisabeth; Guillon, Fabienne; Larré, Colette

    2016-01-01

    Brachypodium distachyon is a suitable plant model for studying temperate cereal crops, such as wheat, barley or rice, and helpful in the study of the grain cell wall. Indeed, the most abundant hemicelluloses that are in the B. distachyon cell wall of grain are (1-3)(1-4)-β-glucans and arabinoxylans, in a ratio similar to those of cereals such as barley or oat. Conversely, these cell walls contain few pectins and xyloglucans. Cell walls play an important role in grain physiology. The modifications of cell wall polysaccharides that occur during grain development and filling are key in the determination of the size and weight of the cereal grains. The mechanisms required for cell wall assembly and remodelling are poorly understood, especially in cereals. To provide a better understanding of these processes, we purified the cell wall at three developmental stages of the B. distachyon grain. The proteins were then extracted, and a quantitative and comparative LC-MS/MS analysis was performed to investigate the protein profile changes during grain development. Over 466 cell wall proteins (CWPs) were identified and classified according to their predicted functions. This work highlights the different proteome profiles that we could relate to the main phases of grain development and to the reorganization of cell wall polysaccharides that occurs during these different developmental stages. These results provide a good springboard to pursue functional validation to better understand the role of CWPs in the assembly and remodelling of the grain cell wall of cereals. PMID:28248231

  11. Ameliorating effects of fennel and cumin extracts on sperm quality and spermatogenic cells apoptosis by inducing weight loss and reducing leptin concentration in diet-induced obese rats.

    PubMed

    Nejatbakhsh, R; Riyahi, S; Farrokhi, A; Rostamkhani, S; Mahmazi, S; Yazdinezhad, A; Kazemi, M; Shokri, S

    2016-11-30

    This study was established a model of obesity to estimate the impact of fennel and cumin as anti-obesity extracts on body weight, body mass index (BMI), food consumption, leptin concentration, sperm quality and testis architecture to determine the reversibility of reproductive function of obese animals. Male rats were randomly assigned to either a normal or high-fat diet for 8 weeks. Then, we divided 56 adult rats into seven groups: control (CO); obesity (OB); fennel 100 and 200 mg/kg; cumin 50 and 100 mg/kg; and fennel 100 mg/kg plus cumin 50 mg/kg. From weeks 9-16, the animals treated extracts by gavages daily. We analysed leptin concentration, sperm quality and apoptosis of testis along with evaluating changes in body weight. Body weight of animals increased 25% at week 8. However, body weight, BMI, leptin concentration and apoptosis indices of OB rats increased at the end of study. However, the relative sperm parameters decreased. Nevertheless, fennel and cumin treatment improved sperm quality, and spermatogenic cells apoptosis following weight loss. Concomitant with weight loss, leptin concentration and food consumption decreased. In conclusion, fennel and cumin as supplements may ameliorate sperm quality of obese animals following weight loss and reduction in leptin concentration.

  12. Susceptibility of human sperm to in situ DNA denaturation is strongly correlated with DNA strand breaks identified by single-cell electrophoresis.

    PubMed

    Aravindan, G R; Bjordahl, J; Jost, L K; Evenson, D P

    1997-10-10

    Susceptibility of mammalian sperm DNA to low pH- or heat-induced denaturation in situ has shown very strong dose-response relationships with animal and human exposure to chemical and physical toxicants and also fertility potential. In this study, 23 human semen samples representing a wide range in percentage (7-86%) of sperm exhibiting abnormally high susceptibility of DNA in situ to denaturation were studied for the integrity of their DNA using alkaline comet assay (single-cell microgel electrophoresis, pH 10.0). The percentage of comets observed for these samples ranged from 5 to 95%; these data correlated strongly with the percentage of sperm with increased DNA denaturability (r = 0.973; P < 0.001). Labeling of 3' ends of nicked DNA sites with 5-bromo-2'-deoxyuridine 5'-triphosphate (BrdUTP) followed by tagging with FITC-BrdUTP monoclonal antibody and flow cytometry also indicated significantly strong correlations of BrdUTP incorporation with both abnormal susceptibility of DNA to denaturation (r = 0.859, P < 0.001) and comet assay (r = 0.812, P < 0.001). The relationship among susceptibility of sperm chromatin to acid denaturation in situ, BrdUTP incorporation, and formation of comets suggests that DNA fragmentation monitored by these assays may have important physiological relevance in terms of sperm quality and fertility potential.

  13. Intracytoplasmic Sperm Injection Using DNA-Fragmented Sperm in Mice Negatively Affects Embryo-Derived Embryonic Stem Cells, Reduces the Fertility of Male Offspring and Induces Heritable Changes in Epialleles

    PubMed Central

    Fernández-González, Raúl; Laguna-Barraza, Ricardo; Pericuesta, Eva; Calero, Antonia; Ramírez, Miguel Ángel; Gutiérrez-Adán, Alfonso

    2014-01-01

    Intracytoplasmic sperm injection (ICSI) in mice using DNA-fragmented sperm (DFS) has been linked to an increased risk of genetic and epigenetic abnormalities both in embryos and offspring. This study examines: whether embryonic stem cells (ESCs) derived from DFS-ICSI embryos reflect the abnormalities observed in the DFS-ICSI progeny; the effect of DFS-ICSI on male fertility; and whether DFS-ICSI induces epigenetic changes that lead to a modified heritable phenotype. DFS-ICSI-produced embryos showed a low potential to generate ESC lines. However, these lines had normal karyotype accompanied by early gene expression alterations, though a normal expression pattern was observed after several passages. The fertility of males in the DFS-ICSI and control groups was compared by mating test. Sperm quantity, vaginal plug and pregnancy rates were significantly lower for the DFS-ICSI-produced males compared to in vivo-produced mice, while the number of females showing resorptions was higher. The epigenetic effects of DFS-ICSI were assessed by analyzing the phenotype rendered by the Axin1Fu allele, a locus that is highly sensitive to epigenetic perturbations. Oocytes were injected with spermatozoa from Axin1Fu/+ mice and the DFS-ICSI-generated embryos were transferred to females. A significantly higher proportion of pups expressed the active kinky-tail epiallele in the DFS-ICSI group than the controls. In conclusion: 1) ESCs cannot be used as a model of DFS-ICSI; 2) DFS-ICSI reduces sperm production and fertility in the male progeny; and 3) DFS-ICSI affects the postnatal expression of a defined epigenetically sensitive allele and this modification may be inherited across generations. PMID:24743851

  14. Sperm Binding to Oviduct Epithelial Cells Enhances TGFB1 and IL10 Expressions in Epithelial Cells as Well as Neutrophils In Vitro: Prostaglandin E2 As a Main Regulator of Anti-Inflammatory Response in the Bovine Oviduct

    PubMed Central

    Yousef, Mohamed Samy; Marey, Mohamed Ali; Hambruch, Nina; Hayakawa, Hiroyuki; Shimizu, Takashi; Hussien, Hassan Ali; Abdel-Razek, Abdel-Razek Khalifa; Pfarrer, Christiane; Miyamoto, Akio

    2016-01-01

    Sperm are allogenic to the female genital tract; however, oviducts provide optimal conditions for survival and capacitation of these non-self cells until fertilization. Recently, we showed that oviduct-conditioned media and prostaglandin E2 (PGE2) suppress sperm phagocytosis by polymorphonuclear neutrophils (PMNs) under physiological conditions. We hypothesized that sperm binding to bovine oviduct epithelial cells (BOECs) could change the local innate immunity via PGE2. As the first step to obtain basic information, sub-confluent BOEC monolayers were co-cultured with swim-up sperm for 2 h. BOECs with viable bound sperm were cultured for an additional 3, 6, 12, or 24 h. Then, we confirmed the impact of the sperm-BOEC binding on both BOECs and PMN gene expression. Immunohistochemistry revealed that BOECs strongly express TGFB1 and IL10 in the oviduct. Sperm binding to BOECs in culture induced the anti-inflammatory cytokines (TGFB1 and IL10) and PGE2 production by BOECs. Exogenous PGE2 in vitro suppressed pro-inflammatory cytokine expression (TNF and IL1B) in BOECs. Moreover, pre-exposure of PMNs to BOEC-conditioned media suppressed the TNF expression, but the BOEC media co-cultured with sperm stimulated PMNs to express TGFB1 and IL10, with increasing PGE2 secretion. Of note, exogenous PGE2 led PMNs in vitro to decrease their TNF expression and increase anti-inflammatory cytokines expression. Our findings strongly suggest that BOECs provide an anti-inflammatory environment under physiological conditions and the sperm-BOEC binding further strengthens this milieu thus suppresses PMNs in the bovine oviduct. PGE2 is likely to drive this stable anti-inflammatory environment in the oviduct. PMID:27662642

  15. Sperm counts and serum follicle-stimulating hormone levels before and after radiotherapy and chemotherapy in men with testicular germ cell cancer

    SciTech Connect

    Berthelsen, J.G.

    1984-02-01

    Sperm counts were low (median, 15 X 10(6) per ejaculate) and serum follicle-stimulating hormone (FSH) levels were moderately elevated (median, 31 IU/l) after unilateral orchiectomy and immediately before radiotherapy and chemotherapy in 34 patients with seminomas and 20 patients with nonseminomatous germ cell tumors. The scattered radiation (0.2 to 1.3 Gray (Gy)) reaching the remaining testicle during radiotherapy caused azoospermia in more than two thirds of the patients. A median of 540 days elapsed after the end of treatment before spermatozoa were again found in semen samples, while a median of 1250 days passed before the pretreatment sperm count was reached. One to 5 years after treatment, sperm counts were still low (median, 6 X 10(6) per ejaculate) and serum FSH was elevated (median, 61 IU/l). The adjuvant chemotherapy given to the 20 patients with nonseminomatous tumors did not appear to affect restitution appreciably.

  16. Effect of Metals on the Development of Hyperactivated Motility by Rabbit Sperm Cells.

    DTIC Science & Technology

    1994-11-01

    Dairy Sci. Vol. 67, pp 1147-1156 (1984). 13. Carreras, A., and Mendoza, C., "Zinc Levels in Seminal Plasma of Fertile and Infertile Men ," Andrologia...cadmium chlorides, used in the study, spanned the concentration range of the metals found in seminal plasma of men exposed to the metals. 9󈧎 The highest...269-275 (1990). 28. Ernst, E., and Bonde, J.P., "Sex Hormone and Epididymal Sperm Parameters in rats Following Sub-Chronic Treatment with Hexavalent

  17. In-situ second harmonic generation by cancer cell targeting ZnO nanocrystals to effect photodynamic action in subcellular space.

    PubMed

    Gu, Bobo; Pliss, Artem; Kuzmin, Andrey N; Baev, Alexander; Ohulchanskyy, Tymish Y; Damasco, Jossana A; Yong, Ken-Tye; Wen, Shuangchun; Prasad, Paras N

    2016-10-01

    This paper introduces the concept of in-situ upconversion of deep penetrating near infrared light via second harmonic generation from ZnO nanocrystals delivered into cells to effect photo activated therapies, such as photodynamic therapy, which usually require activation by visible light with limited penetration through biological tissues. We demonstrated this concept by subcellular activation of a photodynamic therapy drug, Chlorin e6, excited within its strong absorption Soret band by the second harmonic (SH) light, generated at 409 nm by ZnO nanocrystals, which were targeted to cancer cells and internalized through the folate-receptor mediated endocytosis. By a combination of theoretical modeling and experimental measurements, we show that SH light, generated in-situ by ZnO nanocrystals significantly contributes to activation of photosensitizer, leading to cell death through both apoptotic and necrotic pathways initiated in the cytoplasm. This targeted photodynamic action was studied using label-free Coherent Anti-Stokes Raman Scattering imaging of the treated cells to monitor changes in the distribution of native cellular proteins and lipids. We found that initiation of photodynamic therapy with upconverted light led to global reduction in the intracellular concentration of macromolecules, likely due to suppression of proteins and lipids synthesis, which could be considered as a real-time indicator of cellular damage from photodynamic treatment. In prospective applications this in-situ photon upconversion could be further extended using ZnO nanocrystals surface functionalized with a specific organelle targeting group, provided a powerful approach to identify and consequently maximize a cellular response to phototherapy, selectively initiated in a specific cellular organelle.

  18. Regulation of gene expression and subcellular protein distribution in MLO-Y4 osteocytic cells by lysophosphatidic acid: Relevance to dendrite outgrowth.

    SciTech Connect

    Waters, Katrina M.; Jacobs, Jon M.; Gritsenko, Marina A.; Karin, Norman J.

    2011-02-26

    Osteoblastic and osteocytic cells are highly responsive to the lipid growth factor lysophosphatidic acid (LPA) but the mechanisms by which LPA alters bone cell functions are largely unknown. A major effect of LPA on osteocytic cells is the stimulation of dendrite membrane outgrowth, a process that we predicted to require changes in gene expression and protein distribution. We employed DNA microarrays for global transcriptional profiling of MLO-Y4 osteocytic cells grown for 6 and 24h in the presence or absence of LPA. We identified 932 transcripts that displayed statistically significant changes in abundance of at least 1.25-fold in response to LPA treatment. Gene ontology (GO) analysis revealed that the regulated gene products were linked to diverse cellular processes, including DNA repair, response to unfolded protein, ossification, protein-RNA complex assembly, and amine biosynthesis. Gene products associated with the regulation of actin microfilament dynamics displayed the most robust expression changes, and LPA-induced dendritogenesis in vitro was blocked by the stress fiber inhibitor cytochalasin D. Mass spectrometry-based proteomic analysis of MLO-Y4 cells revealed significant LPA-induced changes in the abundance of 284 proteins at 6h and 844 proteins at 24h. GO analysis of the proteomic data linked the effects of LPA to cell processes that control of protein distribution and membrane outgrowth, including protein localization, protein complex assembly, Golgi vesicle transport, cytoskeleton-dependent transport, and membrane invagination/endocytosis. Dendrites were isolated from LPA-treated MLO-Y4 cells and subjected to proteomic analysis to quantitatively assess the subcellular distribution of proteins. Sets of 129 and 36 proteins were enriched in the dendrite fraction as compared to whole cells after 6h and 24h of LPA exposure, respectively. Protein markers indicated that membranous organelles were largely excluded from the dendrites. Highly represented among

  19. Effects of dilution and centrifugation on the survival of spermatozoa and the structure of motile sperm cell subpopulations in refrigerated Catalonian donkey semen.

    PubMed

    Miró, J; Taberner, E; Rivera, M; Peña, A; Medrano, A; Rigau, T; Peñalba, A

    2009-11-01

    The aim of this work was to study the effects of dilution and centrifugation (i.e., two methods of reducing the influence of the seminal plasma) on the survival of spermatozoa and the structure of motile sperm cell subpopulations in refrigerated Catalonian donkey (Equus asinus) semen. Fifty ejaculates from nine Catalonian jackasses were collected. Gel-free semen was diluted 1:1, 1:5 or 1:10 with Kenney extender. Another sample of semen was diluted 1:5, centrifuged, and then resuspended with Kenney extender until a final dilution of 25x10(6) sperm/ml was achieved (C). After 24 h, 48 h or 72 h of refrigerated storage at 5 degrees C, aliquots of these semen samples were incubated at 37 degrees C for 5 min. The percentage of viable sperm was determined by staining with eosin-nigrosin. The motility characteristics of the spermatozoa were examined using the CASA system (Microptic, Barcelona, Spain). At 24h, more surviving spermatozoa were seen in the more diluted and in the centrifuged semen samples (1:1 48.71%; 1:5 56.58%, 1:10 62.65%; C 72.40%). These differences were maintained at 48 h (1:1 34.31%, 1:5 40.56%, 1:10 48.52%, C 66.30%). After 72 h, only the C samples showed a survival rate of above 25%. The four known donkey motile sperm subpopulations were maintained by refrigeration. However, the percentage of motile sperms in each subpopulation changed with dilution. Only the centrifuged samples, and only at 24h, showed exactly the same motile sperm subpopulation proportions as recorded for fresh sperm. However, the 1:10 dilutions at 24 and 48 h, and the centrifuged semen at 48 h, showed few variations compared to fresh sperm. These results show that the elimination of seminal plasma increases the survival of spermatozoa and the maintenance of motility patterns. The initial sperm concentration had a significant (P<0.05) influence on centrifugation efficacy, but did not influence the number of spermatozoa damaged by centrifugation. In contrast, the percentage of live

  20. Fluorescent probe based subcellular distribution of Cu(II) ions in living electrotrophs isolated from Cu(II)-reduced biocathodes of microbial fuel cells.

    PubMed

    Tao, Ye; Xue, Hua; Huang, Liping; Zhou, Peng; Yang, Wei; Quan, Xie; Yuan, Jinxiu

    2017-02-01

    Based on the four indigenous electrotrophs (Stenotrophomonas maltophilia JY1, Citrobacter sp. JY3, Pseudomonas aeruginosa JY5 and Stenotrophomonas sp. JY6) isolated from well adapted Cu(II)-reduced biocathodes of microbial fuel cells (MFCs), a rhodamine based Cu(II) fluorescent probe was used to imaginably and quantitatively track subcellular Cu(II) ions in these electrotrophs. Cathodic electrons led to more Cu(II) ions (14.3-30.1%) in the intracellular sites at operation time of 2-3h with Cu(II) removal rates of 2.90-3.64mg/Lh whereas the absence of cathodic electrons prolonged the appearance of more Cu(II) ions (16.6-22.5%) to 5h with Cu(II) removal rates of 1.96-2.28mg/Lh. This study illustrates that cathodic electrons directed more Cu(II) ions for quicker entrance into the electrotrophic cytoplasm, and gives an alternative approach for developing imaging and functionally tracking Cu(II) ions in the electrotrophs of MFCs.

  1. Phosphorylation and subcellular localization of p27Kip1 regulated by hydrogen peroxide modulation in cancer cells.

    PubMed

    Ibañez, Irene L; Bracalente, Candelaria; Notcovich, Cintia; Tropper, Ivanna; Molinari, Beatriz L; Policastro, Lucía L; Durán, Hebe

    2012-01-01

    The Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H(2)O(2)) in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p) at serine 10 (S10) and at threonine 198 (T198) because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H(2)O(2) removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H(2)O(2) (0.1 µM) to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H(2)O(2) scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization of p27

  2. Phosphorylation and Subcellular Localization of p27Kip1 Regulated by Hydrogen Peroxide Modulation in Cancer Cells

    PubMed Central

    Ibañez, Irene L.; Bracalente, Candelaria; Notcovich, Cintia; Tropper, Ivanna; Molinari, Beatriz L.; Policastro, Lucía L.; Durán, Hebe

    2012-01-01

    The Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H2O2) in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p) at serine 10 (S10) and at threonine 198 (T198) because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H2O2 removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H2O2 (0.1 µM) to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H2O2 scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization of p27Kip1. PMID

  3. The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium

    PubMed Central

    1989-01-01

    Studies of the developing trophectoderm in the mouse embryo have shown that extensive cellular remodeling occurs during epithelial formation. In this investigation, confocal immunofluorescence microscopy is used to examine the three-dimensional changes in cellular architecture that take place during the polarization of a terminally differentiated epithelial cell line. Madin-Darby canine kidney cells were plated at a low density on permeable filter supports. Antibodies that specifically recognize components of the tight junction, adherens junction, microtubules, centrosomes, and the Golgi complex were used to study the spatial remodeling of the cytoarchitecture during the formation of the polarized cell layer. The immunofluorescence data were correlated with establishment of functional tight junctions as measured by transepithelial resistance and back-exchange of the cell surface, labeled with metabolites of the fluorescent lipid analogue N-(7-[4- nitrobenzo-2-oxa-1,3-diazole]) aminocaproyl sphingosine. 1 d after plating, single cells had microtubules, radiating from a broad region, that contained the centrosomes and the Golgi complex. 2 d after plating, the cells had grown to confluence and had formed functional tight junctions close to the substratum. The centrioles had split and no longer organized the microtubules which were running above and below the nucleus. The Golgi complex had spread around the nucleus. By the fifth day after plating, the final polarized state had been achieved. The junctional complex had moved greater than 10 microns upward from its basal location. The centrioles were together below the apical membrane, and the Golgi complex formed a ribbon-like convoluted structure located in the apical region above the nucleus. The microtubules were organized in an apical web and in longitudinal microtubule bundles in the apical-basal axis of the columnar cell. The longitudinal microtubules were arranged with their minus ends spread over the apical

  4. Sperm competition leads to functional adaptations in avian testes to maximize sperm quantity and quality.

    PubMed

    Lüpold, Stefan; Wistuba, Joachim; Damm, Oliver S; Rivers, James W; Birkhead, Tim R

    2011-05-01

    The outcome of sperm competition (i.e. competition for fertilization between ejaculates from different males) is primarily determined by the relative number and quality of rival sperm. Therefore, the testes are under strong selection to maximize both sperm number and quality, which are likely to result in trade-offs in the process of spermatogenesis (e.g. between the rate of spermatogenesis and sperm length or sperm energetics). Comparative studies have shown positive associations between the level of sperm competition and both relative testis size and the proportion of seminiferous (sperm-producing) tissue within the testes. However, it is unknown how the seminiferous tissue itself or the process of spermatogenesis might evolve in response to sperm competition. Therefore, we quantified the different germ cell types and Sertoli cells (SC) in testes to assess the efficiency of sperm production and its associations with sperm length and mating system across 10 species of New World Blackbirds (Icteridae) that show marked variation in sperm length and sperm competition level. We found that species under strong sperm competition generate more round spermatids (RS)/spermatogonium and have SC that support a greater number of germ cells, both of which are likely to increase the maximum sperm output. However, fewer of the RS appeared to elongate to mature spermatozoa in these species, which might be the result of selection for discarding spermatids with undesirable characteristics as they develop. Our results suggest that, in addition to overall size and gross morphology, testes have also evolved functional adaptations to maximize sperm quantity and quality.

  5. The compartmentalized vessel: The bacterial cell as a model for subcellular organization (a tale of two studies).

    PubMed

    Amster-Choder, Orna

    2011-03-01

    The traditional view of bacterial cells as non-compartmentalized, which is based on the lack of membrane-engulfed organelles, is currently being reassessed. Many studies in recent years led to the realization that bacteria have an intricate internal organization that is vital for various cellular processes. Specifically, various machineries were shown to localize to the poles of rod-shaped bacteria. We have recently shown that the control center of the PTS system, which governs carbon uptake and metabolism, localizes to the poles of E. coli cells. Notably, the machinery that controls bacterial taxis along chemical gradients (chemotaxis) has a similar localization pattern. The fact that the two systems need to communicate in order to generate an optimal metabolic response suggests that their similar spatial organization is not a coincidence. Rather, due to their special characteristics, the poles may function as hubs for signaling systems to allow for efficient crosstalk between different pathways in order to improve coordination of their actions.The regulatory mechanisms that underlie the spatial and temporal organization of microbial cells are largely unknown. Thus far, these mechanisms were believed to rely on embedded features of the localized proteins. In another study, we have recently shown that mRNAs are capable of migrating to particular domains in the bacterial cell where their protein products are required. In contrast to the view that transcription and translation are coupled in bacteria, localization of bacterial transcripts may occur in a translation-independent manner. Hence, it seems that the mechanistic basis for separating transcription and translation is more primitive than assumed up until now. We propose that bacteria synthesize proteins either by a transcription-translation coupled mechanism or by transporting mRNAs away from the transcription apparatus. Obviously, eukaryotic cells rely on the latter mechanism. Hence, the capacity of

  6. Direct observation of internalization and ROS generation of amyloid β-peptide in neuronal cells at subcellular resolution.

    PubMed

    Jiao, Yong; Zhang, Yi; Wei, Yibin; Liu, Zhiwei; An, Wenting; Guo, Maolin

    2012-11-05

    Seeing in many colors: Confocal images acquired using fluorescently labeled amyloid β-peptide revealed its efficient internalization by endocytosis into endosomes/lysosomes of human neuronal cells with a small portion reaching mitochondria, inducing marked cellular and mitochondrial reactive oxygen species production.

  7. The application of alkaline lysis and pressure cycling technology in the differential extraction of DNA from sperm and epithelial cells recovered from cotton swabs.

    PubMed

    Nori, Deepthi V; McCord, Bruce R

    2015-09-01

    This study reports the development of a two-step protocol using pressure cycling technology (PCT) and alkaline lysis for differential extraction of DNA from mixtures of sperm and vaginal epithelial cells recovered from cotton swabs. In controlled experiments, in which equal quantities of sperm and female epithelial cells were added to cotton swabs, 5 min of pressure pulsing in the presence of 0.4 M NaOH resulted in 104 ± 6% recovery of female epithelial DNA present on the swab. Following the pressure treatment, exposing the swabs to a second 5-min alkaline treatment at 95 °C without pressure resulted in the selective recovery of 69 ± 6% of the sperm DNA. The recovery of the vaginal epithelia and sperm DNA was optimized by examining the effect of sodium hydroxide concentration, incubation temperature, and time. Following the alkaline lysis steps, the samples were neutralized with 2 M Tris (pH 7.5) and purified with phenol-chloroform-isoamyl alcohol to permit downstream analysis. The total processing time to remove both fractions from the swab was less than 20 min. Short tandem repeat (STR) analysis of these fractions obtained from PCT treatment and alkaline lysis generated clean profiles of female epithelial DNA and male sperm DNA for 1:1 mixtures of female and male cells and predominant male profiles for mixtures up to 5:1 female to male cells. By reducing the time and increasing the recovery of DNA from cotton swabs, this new method presents a novel and potentially useful procedure for forensic differential extractions.

  8. Subcellular proteomics in neuroscience.

    PubMed

    Li, Ka Wan; Smit, August B

    2008-05-01

    The brain is the most complex and dynamically organized organ of the human body, with a high degree of computation capability enabling the execution of a wide spectrum of physiological processes and behaviors. In the past decades a large number of genomics studies have been undertaken to investigate brain function and brain disorders, but despite these efforts many of the underlying molecular mechanisms still remain largely unknown. The implementation of mass spectrometry based quantitative proteomics in recent years enabled to tap into condition-specific protein trafficking and protein interaction that are the key to organelle proteome (dys)function. The technology for neuroproteomics is still evolving; currently there are no standardized protocols. In this review we describe the most commonly used methods to prepare brain subcellular fractions suitable for proteomics analysis, and highlight the various approaches for quantitative neuroproteomics.

  9. The inflammatory mediator leukotriene D{sub 4} induces subcellular β-catenin translocation and migration of colon cancer cells

    SciTech Connect

    Salim, Tavga; Sand-Dejmek, Janna; Sjölander, Anita

    2014-02-15

    The abnormal activation of the Wnt/β-catenin pathway frequently occurs in colorectal cancer. The nuclear translocation of β-catenin activates the transcription of target genes that promote cell proliferation, survival, and invasion. The pro-inflammatory mediator leukotriene D{sub 4} (LTD{sub 4}) exerts its effects through the CysLT{sub 1} receptor. We previously reported an upregulation of CysLT{sub 1}R in patients with colon cancer, suggesting the importance of leukotrienes in colon cancer. The aim of this study was to investigate the impact of LTD{sub 4} on Wnt/β-catenin signaling and its effects on proliferation and migration of colon cancer cells. LTD{sub 4} stimulation led to an increase in β-catenin expression, β-catenin nuclear translocation and the subsequent transcription of MYC and CCND1. Furthermore, LTD{sub 4} significantly reduced the expression of E-cadherin and β-catenin at the plasma membrane and increased the migration and proliferation of HCT116 colon cancer cells. The effects of LTD{sub 4} can be blocked by the inhibition of CysLT{sub 1}R. Furthermore, LTD{sub 4} induced the inhibition of glycogen synthase kinase 3 (GSK)-3β activity, indicating a crosstalk between the G-protein-coupled receptor CysLT{sub 1} and the Wnt/β-catenin pathway. In conclusion, LTD{sub 4}, which can be secreted from macrophages and leukocytes in the tumor microenvironment, induces β-catenin translocation and the activation of β-catenin target genes, resulting in the increased proliferation and migration of colon cancer cells. - Highlights: • Leukotriene D{sub 4} (LTD{sub 4}) lowers membrane β-catenin but increases nuclear β-catenin levels in colon cancer cells. • In agreement, LTD{sub 4} triggers inactivation of GSK-3β, activation of TCF/LEF and increased expression of Cyclin D1 and c-Myc. • LTD{sub 4} also caused a significant reduction in the expression of E-cadherin and an increased migration of colon cancer cells.

  10. Population imaging at subcellular resolution supports specific and local inhibition by granule cells in the olfactory bulb

    PubMed Central

    Wienisch, Martin; Murthy, Venkatesh N.

    2016-01-01

    Information processing in early sensory regions is modulated by a diverse range of inhibitory interneurons. We sought to elucidate the role of olfactory bulb interneurons called granule cells (GCs) in odor processing by imaging the activity of hundreds of these cells simultaneously in mice. Odor responses in GCs were temporally diverse and spatially disperse, with some degree of non-random, modular organization. The overall sparseness of activation of GCs was highly correlated with the extent of glomerular activation by odor stimuli. Increasing concentrations of single odorants led to proportionately larger population activity, but some individual GCs had non-monotonic relations to concentration due to local inhibitory interactions. Individual dendritic segments could sometimes respond independently to odors, revealing their capacity for compartmentalized signaling in vivo. Collectively, the response properties of GCs point to their role in specific and local processing, rather than global operations such as response normalization proposed for other interneurons. PMID:27388949

  11. Mating behavior and the evolution of sperm design

    PubMed Central

    Schärer, Lukas; Littlewood, D. Timothy J.; Waeschenbach, Andrea; Yoshida, Wataru; Vizoso, Dita B.

    2011-01-01

    Sperm are the most diverse of all animal cell types, and much of the diversity in sperm design is thought to reflect adaptations to the highly variable conditions under which sperm function and compete to achieve fertilization. Recent work has shown that these conditions often evolve rapidly as a consequence of multiple mating, suggesting a role for sexual selection and sexual conflict in the evolution of sperm design. However, very little of the striking diversity in sperm design is understood functionally, particularly in internally fertilizing organisms. We use phylogenetic comparative analyses covering 16 species of the hermaphroditic flatworm genus Macrostomum to show that a complex sperm design is associated with reciprocal mating and that this complexity is lost secondarily when hypodermic insemination—sperm injection through the epidermis—evolves. Specifically, the complex sperm design, which includes stiff lateral bristles, is likely a male persistence trait associated with sexual conflicts over the fate of received ejaculates and linked to female resistance traits, namely an intriguing postcopulatory sucking behavior and a thickened epithelium of the sperm-receiving organ. Our results suggest that the interactions between sperm donor, sperm, and sperm recipient can change drastically when hypodermic insemination evolves, involving convergent evolution of a needle-like copulatory organ, a simpler sperm design, and a simpler female genital morphology. Our study documents that a shift in the mating behavior may alter fundamentally the conditions under which sperm compete and thereby lead to a drastic change in sperm design. PMID:21220334

  12. Establishment of a human cell line stably overexpressing mouse Nip45 and characterization of Nip45 subcellular localization

    SciTech Connect

    Hashiguchi, Kohtaro; Ozaki, Masumi; Kuraoka, Isao; Saitoh, Hisato

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer A human cell line expressing a mouse Nip45 has facilitated Nip45 analysis. Black-Right-Pointing-Pointer Nip45 does not effectively inhibit polySUMOylation in vivo. Black-Right-Pointing-Pointer Nip45 interacts directly with SUMO and SUMO chains. Black-Right-Pointing-Pointer Nip45 accumulates at PML bodies in response to proteasome inhibition. -- Abstract: The nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 2 interacting protein, Nfatc2ip (Nip45), has been implicated as a crucial coordinator of the immune response and of cellular differentiation in humans and mice, and contains SUMO-like domains in its C-terminal region. However, the significance of its N-terminal region and its correlation to the SUMO modification pathway remain largely uncharacterized. In this study, a human cultured cell line was established, in which FLAG-tagged mouse Nip45 (FLAG-mNip45) was stably overexpressed. Under standard, non-stressful conditions, we detected FLAG-mNip45 diffusely distributed in the nucleus. Intriguingly, proteasome inhibition by MG132 caused FLAG-mNip45, together with SUMOylated proteins, to localize in nuclear domains associated with promyelocytic leukemia protein. Finally, using an in vitro binding assay, we showed interaction of the N-terminal region of mNip45 with both free SUMO-3 and SUMO-3 chains, indicating that Nip45 may, in part, exert its function via interaction with SUMO/SUMOylated proteins. Taken together, our study provides novel information on a poorly characterized mammalian protein and suggests that our newly established cell line will be useful for elucidating the physiological role of Nip45.

  13. Trimethyltin retinopathy: relationship of subcellular response to neuronal subspecialization

    SciTech Connect

    Bouldin, T.W.; Goines, N.D.; Krigman, M.R.

    1984-03-01

    Retinal neurons from rats acutely intoxicated with trimethyltin (TMT) were examined by light and electron microscopy to determine if there is a relationship between the subcellular response of a neuron to TMT and its morphologic subspecialization. Subcellular pathologic alterations were present in neurons from all three cellular layers of the sensory retina. However, the type and degree of subcellular response varied among the highly subspecialized neurons of the different retinal layers. Clusters of dense-cored vesicles and tubules were mainly limited to neurons of the ganglion-cell layer, large accumulations of dense bodies were mainly limited to neurons of the inner nuclear layer, and neuronal necrosis was mainly limited to the photoreceptor cells. The inner segment of the photoreceptor cell shared with the perikaryon of more conventional neurons a special vulnerability to TMT cytotoxicity. Our results suggest that the subspecialization of neurons affects the type and the degree of subcellular response to TMT.

  14. Characterisation of Caenorhabditis elegans sperm transcriptome and proteome

    PubMed Central

    2014-01-01

    Background Although sperm is transcriptionally and translationally quiescent, complex populations of RNAs, including mRNAs and non-coding RNAs, exist in sperm. Previous microarray analysis of germ cell mutants identified hundreds of sperm genes in Caenorhabditis elegans. To take a more comprehensive view on C. elegans sperm genes, here, we isolate highly pure sperm cells and employ high-throughput technologies to obtain sperm transcriptome and proteome. Results First, sperm transcriptome consists of considerable amounts of non-coding RNAs, many of which have not been annotated and may play functional roles during spermatogenesis. Second, apart from kinases/phosphatases as previously reported, ion binding proteins are also enriched in sperm, underlying the crucial roles of intracellular ions in post-translational regulation in sperm. Third, while the majority of sperm genes/proteins have low abundance, a small number of sperm genes/proteins are hugely enriched in sperm, implying that sperm only rely on a small set of proteins for post-translational regulation. Lastly, by extensive RNAi screening of sperm enriched genes, we identified a few genes that control fertility. Our further analysis reveals a tight correlation between sperm transcriptome and sperm small RNAome, suggesting that the endogenous siRNAs strongly repress sperm genes. This leads to an idea that the inefficient RNAi screening of sperm genes, a phenomenon currently with unknown causes, might result from the competition between the endogenous RNAi pathway and the exogenous RNAi pathway. Conclusions Together, the obtained sperm transcriptome and proteome serve as valuable resources to systematically study spermatogenesis in C. elegans. PMID:24581041

  15. Signaling in Sperm: More Different than Similar.

    PubMed

    Kaupp, U B; Strünker, T

    2017-02-01

    For a given sensory cell type, signaling motifs are rather uniform across phyla. By contrast, sperm from different species use diverse repertoires of sperm-specific signaling molecules and even closely related protein isoforms feature different properties and serve different functions. This surprising diversity has consequences for strategies in fertilization research and it will take some time to get the big picture. We discuss the function of receptors, ion channels, and exchangers embedded in cellular pathways from different sperm species.

  16. Sperm traits in farmed and wild Atlantic salmon Salmo salar.

    PubMed

    Camarillo-Sepulveda, N; Hamoutene, D; Lush, L; Burt, K; Volkoff, H; Fleming, I A

    2016-02-01

    Differences in sperm metabolism and morphology between wild and non-local farmed Atlantic salmon Salmo salar were assessed by measuring metabolic enzyme activities and length of sperm flagella. No differences were observed between wild and farmed S. salar sperm with regards to cell counts or any of the biochemical variables assessed. Flagella of sperm cells were significantly longer in wild than farmed S. salar; however, this did not result in higher energy levels or different fertilization rates.

  17. Cryopreservation and in vitro culture of primary cell types from lung tissue of a stranded pygmy sperm whale (Kogia breviceps).

    PubMed

    Annalaura Mancia; Spyropoulos, Demetri D; McFee, Wayne E; Newton, Danforth A; Baatz, John E

    2012-01-01

    Current models for in vitro studies of tissue function and physiology, including responses to hypoxia or environmental toxins, are limited and rely heavily on standard 2-dimensional (2-D) cultures with immortalized murine or human cell lines. To develop a new more powerful model system, we have pursued methods to establish and expand cultures of primary lung cell types and reconstituted tissues from marine mammals. What little is known about the physiology of the deep-sea diving pygmy sperm whale (PSW), Kogia breviceps, comes primarily from stranding events that occur along the coast of the southeastern United States. Thus, development of a method for preserving live tissues and retrieving live cells from deceased stranded individuals was initiated. This report documents successful cryopreservation of PSW lung tissue. We established in vitro cultures of primary lung cell types from tissue fragments that had been cryopreserved several months earlier at the stranding event. Dissociation of cryopreserved lung tissues readily provides a variety of primary cell types that, to varying degrees, can be expanded and further studied/manipulated in cell culture. In addition, PSW-specific molecular markers have been developed that permitted the monitoring of fibroblast, alveolar type II, and vascular endothelial cell types. Reconstitution of 3-D cultures of lung tissues with these cell types is now underway. This novel system may facilitate the development of rare or disease-specific lung tissue models (e.g., to test causes of PSW stranding events and lead to improved treatments for pulmonary hypertension or reperfusion injury in humans). Also, the establishment of a "living" tissue bank biorepository for rare/endangered species could serve multiple purposes as surrogates for freshly isolated samples.

  18. Prognostic significance of p62/SQSTM1 subcellular localization and LC3B in oral squamous cell carcinoma

    PubMed Central

    Liu, J-L; Chen, F-F; Lung, J; Lo, C-H; Lee, F-H; Lu, Y-C; Hung, C-H

    2014-01-01

    Background: Autophagy is a programmed cell survival mechanism that has a key role in both physiologic and pathologic conditions. The relationship between autophagy and cancer is complex because autophagy can act as either a tumour suppressor or as a tumour promoter. The role of autophagy in oral squamous cell carcinoma (OSCC) is controversial. Several studies have claimed that either a high or low expression of autophagy-related proteins was associated with poor prognosis of OSCCs. The aims of the study were to compare autophagy in OSCCs, verrucous hyperplasias, and normal oral mucosas, and to inspect the prognostic role of autophagy in OSCCs. Methods: We used the autophagosome marker, LC3B, and autophagy flux marker, p62/SQSTM1 (p62), by using immunohistochemistry, and examined p62 mRNA by RNA in situ hybridization, to evaluate autophagy in 195 OSCCs, 47 verrucous hyperplasias, and 37 normal oral mucosas. The prognostic roles of LC3B and p62 protein expressions in OSCCs were investigated. Results: We discovered that the normal oral mucosa exhibited limited LC3B punctae and weak cytoplasmic p62 staining, whereas the OSCCs exhibited a marked increase in LC3B punctae and cytoplasmic p62 expression. The expression pattern of LC3B and cytoplasmic p62 of the verrucous hyperplasias were between normal oral mucosas and OSCCs. The normal oral mucosas, verrucous hyperplasias, and OSCCs presented no differences in nuclear p62 expression and the p62 mRNA level. p62 mRNA expression was elevated in a minority of cases. High p62 mRNA expression was associated with high p62 protein expression in the cytoplasm. Increased LC3B punctae, high cytoplasmic p62, and low nuclear p62 expressions in OSCCs were associated with aggressive clinicopathologic features and unfavourable prognosis. In addition, low nuclear p62 expression was an independent prognostic factor for overall and disease-specific survival rates. Furthermore, we disclosed that high cytoplasmic p62 expression accompanied

  19. Cytometry of mammalian sperm

    SciTech Connect

    Gledhill, B.L.

    1983-10-11

    Male germ cells respond dramatically to a variety of insults and are important reproductive dosimeters. Semen analyses are very useful in studies on the effects of drugs, chemicals, and environmental hazards on testicular function, male fertility and heritable germinal mutations. The accessibility of male cells makes them well suited for analytical cytology. We might automate the process of determining sperm morphology but should not do so solely for increased speed. Rather, richer tangible benefits will derive from cytometric evaluation through increased sensitivity, reduced subjectivity, standardization between investigators and laboratories, enhanced archival systems, and the benefits of easily exchanged standardized data. Inroads on the standardization of assays for motility and functional integrity are being made. Flow cytometric analysis of total DNA content of individual sperm is an insensitive means to detect exposure to reproductive toxins because of the small size and low frequency of the DNA content errors. Flow cytometry can be applied to determine the proportions of X- and Y-sperm in semen samples.

  20. Sperm macrocephaly syndrome in a patient without AURKC mutations and with a history of recurrent miscarriage.

    PubMed

    Molinari, Emanuela; Mirabelli, Marzia; Raimondo, Stefania; Brussino, Alessandro; Gennarelli, Gianluca; Bongioanni, Francesca; Revelli, Alberto

    2013-02-01

    This paper reports a case of recurrent miscarriage in a patient affected by a variant phenotype of sperm macrocephaly syndrome (SMS). SMS is usually related to specific sperm characteristics (large head, multiple tail) and homozygous mutations in the aurora kinase C gene (AURKC). However, the present case observed large-headed spermatozoa with no flagellar abnormalities and no mutations detectable by AURKC sequencing. Furthermore, the patient had repeatedly conceived by intracytoplasmic sperm injection, but pregnancy always aborted. This study performed morphological analysis (Papanicolau staining), annexin V/propidium iodide staining, sperm chromatin structure assay (SCSA), fluorescence in-situ hybridization (FISH) and transmission electron microscopy. This study observed large-headed, mono-tailed, mono-centriolar spermatozoa characterized by abnormal chromatin and swollen mitochondria. SCSA revealed a high ratio of late apoptotic cells with fairly intact amount of DNA. The FISH analysis showed 100% disomy rate. As far as is known, this is the first study to include gene sequencing, TEM, cytogenetic analysis and sperm DNA fragmentation in a case of SMS and also to report recurrent miscarriage related to this specific condition. SMS may be associated with important abnormalities of the sperm subcellular structure and with disomy even in the absence of mutations in the AURKC coding sequence. Sperm macrocephaly syndrome (SMS) is a rare condition that affects spermatozoa and is related to infertility. It is characterized by a specific phenotype of large-headed, multi-tailed spermatozoa with an abnormal chromosomal status. A very few pregnancies have been obtained so far in SMS patients by means of IVF procedures. We present a case of SMS that differs from the classical syndrome as we observed large-headed spermatozoa without tail abnormalities. The affected patient had achieved three pregnancies following IVF, but all aborted. We carried out a detailed examination of

  1. ADM-1, a protein with metalloprotease- and disintegrin-like domains, is expressed in syncytial organs, sperm, and sheath cells of sensory organs in Caenorhabditis elegans.

    PubMed Central

    Podbilewicz, B

    1996-01-01

    A search was carried out for homologues of possible fusogenic proteins to study their function in a genetically tractable animal. The isolation, molecular, and cellular characterization of the Caenorhabditis elegans adm-1 gene (a disintegrin and metalloprotease domain) are described. A glycoprotein analogous to viral fusion proteins has been identified on the surface of guinea pig sperm (PH-30/fertilin) and is implicated in sperm-egg fusion. adm-1 is the first reported invertebrate gene related to PH-30 and a family of proteins containing snake venom disintegrin- and metalloprotease-like domains. ADM-1 shows a domain organization identical to PH-30. It contains prepro, metalloprotease, disintegrin, cysteine rich with putative fusion peptide, epidermal growth factor-like repeat, transmembrane, and cytoplasmic domains. Antibodies which recognize ADM-1 protein in immunoblots were generated. Using immunofluorescence and in situ hybridization, the products of adm-1 have been detected in specific cells during different stages of development. The localization of ADM-1 to the plasma membrane of embryonic cells and to the sheath cells of sensory organs suggests a function in cell adhesion. ADM-1 expression in the hypodermis, pharynx, vulva, and mature sperm is consistent with a putative role in somatic and gamete cell fusions. Images PMID:8970152

  2. Intracellular pH in sperm physiology.

    PubMed

    Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L; Darszon, Alberto

    2014-08-01

    Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca(2+) channel; Slo3, a K(+) channel; the sperm-specific Na(+)/H(+) exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction.

  3. Age and markers of Leydig cell function, but not of Sertoli cell function predict the success of sperm retrieval in adolescents and adults with Klinefelter's syndrome.

    PubMed

    Rohayem, J; Fricke, R; Czeloth, K; Mallidis, C; Wistuba, J; Krallmann, C; Zitzmann, M; Kliesch, S

    2015-09-01

    Microsurgical testicular sperm extraction (mTESE), combined with intracytoplasmic sperm injection (ICSI) represents a chance for azoospermic men with Klinefelter's syndrome (KS) to father children. The objective of this study was to identify predictive factors for the success of mTESE from adolescents and adults with KS. The clinical data of 50 late pubertal adolescents (13-19 years) and 85 adult patients (20-61 years) with non-mosaic KS, who underwent mTESE, were analysed with respect to factors, potentially predictive of active spermatogenesis; specifically a history of cryptorchidism, age, testicular volumes, serum levels of LH, FSH, testosterone (T) and estradiol at the time of surgery. Inhibin B, AMH and INSL3 were additionally analysed in the adolescents. A younger age and a near-compensated Leydig cell function were associated with higher success of sperm retrieval via mTESE: In adolescents ≥15-19 years, spermatozoa were retrieved in 45%, compared to 31% in adults; in adolescents aged 13-14 years, spermatozoa were collected in only 10%. Adolescents with an LH ≤17.5 U/L, along with a T level ≥7.5 nmol/L had the best success rate (54%), which fell to 44% with higher LH, whereas those with low T (<7.5 nmol/L), irrespective of LH had no sperm retrieval. In adults with T levels above and LH below these thresholds, the success rate was 51%, falling to 19%, if LH was higher. When T was lower than threshold, the rate was 17%. No association between testicular volumes, serum levels of FSH, Inhibin B, AMH, estradiol and mTESE success was found. A history of cryptorchidism was associated with lower retrieval rates. A window of opportunity for an approximate 50% chance to retrieve spermatozoa via mTESE exists for young, late pubertal KS patients between age 15 and young adulthood, when Leydig cell function is at its best. In these cases, referral to a centre of expertise should be considered.

  4. Increasing versatility of the DNA vaccines through modification of the subcellular location of plasmid-encoded antigen expression in the in vivo transfected cells.

    PubMed

    Martinez-Lopez, Alicia; García-Valtanen, Pablo; Ortega-Villaizan, María Del Mar; Chico, Verónica; Medina-Gali, Regla María; Perez, Luis; Coll, Julio; Estepa, Amparo

    2013-01-01

    The route of administration of DNA vaccines can play a key role in the magnitude and quality of the immune response triggered after their administration. DNA vaccines containing the gene of the membrane-anchored glycoprotein (gpG) of the fish rhabdoviruses infectious haematopoietic necrosis virus (IHNV) or viral haematopoietic septicaemia virus (VHSV), perhaps the most effective DNA vaccines generated so far, confer maximum protection when injected intramuscularly in contrast to their low efficacy when injected intraperitoneally. In this work, taking as a model the DNA vaccine against VHSV, we focused on developing a more versatile DNA vaccine capable of inducing protective immunity regardless of the administration route used. For that, we designed two alternative constructs to gpG₁₋₅₀₇ (the wild type membrane-anchored gpG of VHSV) encoding either a soluble (gpG₁₋₄₆₂) or a secreted soluble (gpG(LmPle20-462)) form of the VHSV-gpG. In vivo immunisation/challenge assays showed that only gpG(LmPle20-462) (the secreted soluble form) conferred protective immunity against VHSV lethal challenge via both intramuscular and intraperitoneal injection, being this the first description of a fish viral DNA vaccine that confers protection when administered intraperitoneally. Moreover, this new DNA vaccine construct also conferred protection when administered in the presence of an oil adjuvant suggesting that DNA vaccines against rhabdoviruses could be included in the formulation of current multicomponent-intaperitoneally injectable fish vaccines formulated with an oil adjuvant. On the other hand, a strong recruitment of membrane immunoglobulin expressing B cells, mainly membrane IgT, as well as t-bet expressing T cells, at early times post-immunisation, was specifically observed in the fish immunised with the secreted soluble form of the VHSV-gpG protein; this may indicate that the subcellular location of plasmid-encoded antigen expression in the in vivo

  5. Hypercholesterolemia Impaired Sperm Functionality in Rabbits

    PubMed Central

    Monclus, Maria A.; Cabrillana, Maria E.; Clementi, Marisa A.; Espínola, Leandro S.; Cid Barría, Jose L.; Vincenti, Amanda E.; Santi, Analia G.; Fornés, Miguel W.

    2010-01-01

    Hypercholesterolemia represents a high risk factor for frequent diseases and it has also been associated with poor semen quality that may lead to male infertility. The aim of this study was to analyze semen and sperm function in diet-induced hypercholesterolemic rabbits. Twelve adult White New Zealand male rabbits were fed ad libitum a control diet or a diet supplemented with 0.05% cholesterol. Rabbits under cholesterol-enriched diet significantly increased total cholesterol level in the serum. Semen examination revealed a significant reduction in semen volume and sperm motility in hypercholesterolemic rabbits (HCR). Sperm cell morphology was seriously affected, displaying primarily a “folded head”-head fold along the major axe-, and the presence of cytoplasmic droplet on sperm flagellum. Cholesterol was particularly increased in acrosomal region when detected by filipin probe. The rise in cholesterol concentration in sperm cells was determined quantitatively by Gas chromatographic-mass spectrometric analyses. We also found a reduction of protein tyrosine phosphorylation in sperm incubated under capacitating conditions from HCR. Interestingly, the addition of Protein Kinase A pathway activators -dibutyryl-cyclic AMP and iso-butylmethylxanthine- to the medium restored sperm capacitation. Finally, it was also reported a significant decrease in the percentage of reacted sperm in the presence of progesterone. In conclusion, our data showed that diet-induced hypercholesterolemia adversely affects semen quality and sperm motility, capacitation and acrosomal reaction in rabbits; probably due to an increase in cellular cholesterol content that alters membrane related events. PMID:20976152

  6. Effect of non-sperm cells removal with single-layer colloidal centrifugation on myeloperoxidase concentration in post-thaw equine semen.

    PubMed

    Ponthier, Jérôme; Teague, Sheila R; Franck, Thierry Y; de la Rebière, Geoffroy; Serteyn, Didier D; Brinsko, Steven P; Love, Charles C; Blanchard, Terry L; Varner, Dickson D; Deleuze, Stéfan C

    2013-12-01

    Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils during degranulation or after lysis. Post-thaw semen contains MPO and its concentration is associated with decreased sperm motility. Recently, MPO concentration in post-thaw semen was shown to be associated with the presence of non-sperm cells (NSC). The objective of this study was to evaluate the effect of a single-layer colloidal centrifugation before cryopreservation on NSC and MPO concentrations in equine semen. The experimental design consisted of freezing semen with or without previous centrifugation through two concentrations of single-layer colloid media. Non-sperm cells and MPO concentrations were assessed in pellet and upper layer at each step of the procedure and MPO was detected in cells by immunocytochemistry. Single-layer colloid centrifugation decreased NSC and MPO concentrations in post-thaw semen. The MPO concentration was correlated with concentration of NSC in the upper layer of the supernatant. In post-thaw semen, with or without previous single-layer colloid centrifugation, MPO concentration was correlated with concentration of NSC. Overall, neutrophils were rarely observed and NSC were mainly epithelial cells or cellular debris, as demonstrated by MPO immunocytochemistry. At all steps of the semen processing and cryopreservation, MPO immunostaining was clearly identified only on NSC. In conclusion, our study shows that NSC present in fresh semen release MPO during freezing.

  7. Expression of granulocyte-macrophage colony stimulating factor (GM-CSF) in male germ cells: GM-CSF enhances sperm motility.

    PubMed

    Vilanova, Lourdes T; Rauch, M Cecilia; Mansilla, Alejandra; Zambrano, Angara; Brito, Mónica; Werner, Enrique; Alfaro, Víctor; Cox, José F; Concha, Ilona I

    2003-10-01

    The granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine capable of stimulating proliferation, maturation and function of hematopoietic cells. Receptors for this cytokine are composed of two subunits, alpha and beta, and are expressed on myeloid progenitors and mature mononuclear phagocytes, monocytes, eosinophils and neutrophils, as well as in other nonhematopietic cells. We have recently demonstrated that bull spermatozoa express functional GM-CSF receptors that signal for increased glucose and Vitamin C uptake. In this study, we analyzed the expression of GM-CSF in bovine and human germ cells and its influence in bovine sperm motility. Reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization and immunoblotting analysis demonstrated that adult bovine and human testes expressed GM-CSF. In addition, immunolocalization studies confirmed the presence of GM-CSF in the germ cell line in bovine and human testes. Computer-assisted evaluation of patterns of sperm motility demonstrated that the addition of GM-CSF enhances several parameters of sperm motility in the presence of glucose or fructose substrates.

  8. Ameliorating Effect of Ginseng on Epididymo-Orchitis Inducing Alterations in Sperm Quality and Spermatogenic Cells Apoptosis following Infection by Uropathogenic Escherichia coli in Rats

    PubMed Central

    Eskandari, Mehdi; Jani, Soghra; Kazemi, Mahsa; Zeighami, Habib; Yazdinezhad, Alireza; Mazloomi, Sahar; Shokri, Saeed

    2016-01-01

    Objective Epididymo-orchitis (EO) potentially results in reduced fertility in up to 60% of affected patients. The anti-inflammatory effects of Korean red ginseng (KRG) and its ability to act as an immunoenhancer in parallel with the beneficial effects of this ancient herbal medicine on the reproductive systems of animals and humans led us to evaluate its protective effects against acute EO. Materials and Methods This animal experimental study was conducted in the Department of Anatomical Sciences, Faculty of Medicine, Zanjan University of Medical Sciences (ZUMS), Zanjan, Iran during 2013-2015. We divided 50 Wistar rats into five following groups (n=10 per group): i. Control-intact animals, ii. Vehicle-phosphate buffered saline (PBS) injection into the vas deferens, iii. KRG-an intraperitoneal (IP) injection of KRG, iv. EO-an injection of uropathogenic Escherichia coli (UPEC) strain M39 into the vas defer- ens, and v. EO/ KRG-injections of both UPEC strain M39 and KRG. The treatment lasted seven days. We then evaluated sperm parameters, number of germ cell layers, Johnson’s criteria, germ cell apoptosis, body weight and relative sex organs weight. Results Acute EO increased the relative weight of prostate and seminal vesicles (P≤0.05). It also reduced sperm quality such as total motility, sperm concentration (P≤0.01), and the percentage of normal sperm (P≤0.001). Moreover, acute EO decreased Miller’s (P≤0.05) and Johnsen’s scores and increased apoptotic indexes of spermatogenic cells (P≤0.001). KRG treatment decreased prostate weight gain (P≤0.05) and improved the percentage of sperm with normal morphology, total motility (P≤0.01), and progressive motility (P≤0.05). The apoptotic indexes of spermatogenic cells reduced (P≤0.001), whereas both Johnsen’s (P≤0.01) and Miller’s criteria increased in the KRG-treated EO testis (P≤0.05). Conclusion Consequently, KRG ameliorated the devastating effects of EO on the sperm retrieved from either

  9. Proteomics of ionomycin-induced ascidian sperm reaction: Released and exposed sperm proteins in the ascidian Ciona intestinalis.

    PubMed

    Nakazawa, Shiori; Shirae-Kurabayashi, Maki; Otsuka, Kei; Sawada, Hitoshi

    2015-12-01

    Sperm proteins mediating sperm-egg interaction should be exhibited on the sperm surface, or exposed or released when sperm approach an egg. In ascidians (protochordates), sperm undergo a sperm reaction, characterized by enhanced sperm motility and mitochondrial swelling and shedding on contact with the vitelline coat (VC) or by treatment with Ca(2+) ionophore. Here, proteomic analysis was conducted on sperm exudates and sperm surface proteins using ionomycin-induced sperm reaction and cell-impermeable labeling in Ciona intestinalis type A (C. robusta). In the exudate from sperm treated with ionomycin, membrane proteins including a possible VC receptor CiUrabin were abundant, indicating the release of membranous compartments during sperm reaction. Among the surface proteins XP_009859314.1 (uncharacterized protein exhibiting homology to HrTTSP-1) was most abundant before the sperm reaction, but XP_004227079.1 (unknown Ig superfamily protein) appears to be most abundantly exposed by the sperm reaction. Moreover, proteins containing a notable set of domains, astacin-like metalloprotease domain and thrombospondin type 1 repeat(s), were found in this fraction. Possible roles in fertilization as well as localizations and behaviors of these proteins are discussed.

  10. Sperm motility of externally fertilizing fish and amphibians.

    PubMed

    Browne, R K; Kaurova, S A; Uteshev, V K; Shishova, N V; McGinnity, D; Figiel, C R; Mansour, N; Agney, D; Wu, M; Gakhova, E N; Dzyuba, B; Cosson, J

    2015-01-01

    We review the phylogeny, sperm competition, morphology, physiology, and fertilization environments of the sperm of externally fertilizing fish and amphibians. Increased sperm competition in both fish and anurans generally increases sperm numbers, sperm length, and energy reserves. The difference between the internal osmolarity and iconicity of sperm cells and those of the aquatic medium control the activation, longevity, and velocity of sperm motility. Hypo-osmolarity of the aquatic medium activates the motility of freshwater fish and amphibian sperm and hyperosmolarity activates the motility of marine fish sperm. The average longevity of the motility of marine fish sperm (~550 seconds) was significantly (P < 0.05) greater than that of freshwater fish sperm (~150 seconds), with the longevities of both marine and freshwater fish being significantly (P < 0.05) lower than that of anuran sperm (~4100 seconds). The average velocity of anuran sperm (25 μm/s) was significantly (P < 0.05) lower than that of marine fish (140 μm/s) or freshwater fish (135 μm/s) sperm. The longevity of the sperm of giant salamanders (Cryptobranchoidea) of approximately 600 seconds was greater than that of freshwater fish sperm but much lower than anuran sperm. Our research and information from the literature showed that higher osmolarities promote greater longevity in anuran sperm, and some freshwater fish sperm, and that anuran and cryptobranchid sperm maintained membrane integrity long after the cessation of motility, demonstrating a preferential sharing of energy reserves toward the maintenance of membrane integrity. The maintenance of the membrane integrity of anuran sperm in fresh water for up to 6 hours showed an extremely high osmotic tolerance relative to fish sperm. The very high longevity and osmotic tolerance of anuran sperm and high longevity of cryptobranchid sperm, relative to those of freshwater fish, may reflect the complex fertilization history of amphibian sperm in

  11. Sperm function test

    PubMed Central

    Talwar, Pankaj; Hayatnagarkar, Suryakant

    2015-01-01

    With absolute normal semen analysis parameters it may not be necessary to shift to specialized tests early but in cases with borderline parameters or with history of fertilization failure in past it becomes necessary to do a battery of tests to evaluate different parameters of spermatozoa. Various sperm function tests are proposed and endorsed by different researchers in addition to the routine evaluation of fertility. These tests detect function of a certain part of spermatozoon and give insight on the events in fertilization of the oocyte. The sperms need to get nutrition from the seminal plasma in the form of fructose and citrate (this can be assessed by fructose qualitative and quantitative estimation, citrate estimation). They should be protected from the bad effects of pus cells and reactive oxygen species (ROS) (leukocyte detection test, ROS estimation). Their number should be in sufficient in terms of (count), structure normal to be able to fertilize eggs (semen morphology). Sperms should have intact and functioning membrane to survive harsh environment of vagina and uterine fluids (vitality and hypo-osmotic swelling test), should have good mitochondrial function to be able to provide energy (mitochondrial activity index test). They should also have satisfactory acrosome function to be able to burrow a hole in zona pellucida (acrosome intactness test, zona penetration test). Finally, they should have properly packed DNA in the nucleus to be able to transfer the male genes (nuclear chromatic decondensation test) to the oocyte during fertilization. PMID:26157295

  12. Prudent sperm use by leaf-cutter ant queens

    PubMed Central

    den Boer, Susanne P. A.; Baer, Boris; Dreier, Stephanie; Aron, Serge; Nash, David R.; Boomsma, Jacobus J.

    2009-01-01

    In many species, females store sperm between copulation and egg fertilization, but the consequences of sperm storage and patterns of sperm use for female life history and reproductive success have not been investigated in great detail. In hymenopteran insect societies (ants, bees, wasps), reproduction is usually monopolized by one or relatively few queens, who mate only during a brief period early in life and store sperm for later use. The queens of some ants are particularly long-lived and have the potential to produce millions of offspring during their life. To do so, queens store many sperm cells, and this sperm must remain viable throughout the years of storage. Queens should also be under strong selection to use stored sperm prudently when fertilizing eggs. We used the leaf-cutter ant Atta colombica to investigate the dynamics of sperm use during egg fertilization. We show that queens are able to fertilize close to 100 per cent of the eggs and that the average sperm use per egg is very low, but increases with queen age. The robustness of stored sperm was found to decrease with years of storage, signifying that senescence affects sperm either directly or indirectly via the declining glandular secretions or deteriorating sperm-storage organs. We evaluate our findings with a heuristic model, which suggests that the average queen has sperm for almost 9 years of normal colony development. We discuss the extent to which leaf-cutter ant queens have been able to optimize their sperm expenditure and infer that our observed averages of sperm number, sperm robustness and sperm use are consistent with sperm depletion being a significant cause of mortality of mature colonies of Atta leaf-cutter ants. PMID:19710057

  13. Prudent sperm use by leaf-cutter ant queens.

    PubMed

    den Boer, Susanne P A; Baer, Boris; Dreier, Stephanie; Aron, Serge; Nash, David R; Boomsma, Jacobus J

    2009-11-22

    In many species, females store sperm between copulation and egg fertilization, but the consequences of sperm storage and patterns of sperm use for female life history and reproductive success have not been investigated in great detail. In hymenopteran insect societies (ants, bees, wasps), reproduction is usually monopolized by one or relatively few queens, who mate only during a brief period early in life and store sperm for later use. The queens of some ants are particularly long-lived and have the potential to produce millions of offspring during their life. To do so, queens store many sperm cells, and this sperm must remain viable throughout the years of storage. Queens should also be under strong selection to use stored sperm prudently when fertilizing eggs. We used the leaf-cutter ant Atta colombica to investigate the dynamics of sperm use during egg fertilization. We show that queens are able to fertilize close to 100 per cent of the eggs and that the average sperm use per egg is very low, but increases with queen age. The robustness of stored sperm was found to decrease with years of storage, signifying that senescence affects sperm either directly or indirectly via the declining glandular secretions or deteriorating sperm-storage organs. We evaluate our findings with a heuristic model, which suggests that the average queen has sperm for almost 9 years of normal colony development. We discuss the extent to which leaf-cutter ant queens have been able to optimize their sperm expenditure and infer that our observed averages of sperm number, sperm robustness and sperm use are consistent with sperm depletion being a significant cause of mortality of mature colonies of Atta leaf-cutter ants.

  14. Sugar‐coated sperm: Unraveling the functions of the mammalian sperm glycocalyx

    PubMed Central

    Tecle, Eillen

    2015-01-01

    SUMMARY Mammalian spermatozoa are coated with a thick glycocalyx that is assembled during sperm development, maturation, and upon contact with seminal fluid. The sperm glycocalyx is critical for sperm survival in the female reproductive tract and is modified during capacitation. The complex interplay among the various glycoconjugates generates numerous signaling motifs that may regulate sperm function and, as a result, fertility. Nascent spermatozoa assemble their own glycans while the cells still possess a functional endoplasmic reticulum and Golgi in the seminiferous tubule, but once spermatogenesis is complete, they lose the capacity to produce glycoconjugates de novo. Sperm glycans continue to be modified, during epididymal transit by extracellular glycosidases and glycosyltransferases. Furthermore, epididymal cells secrete glycoconjugates (glycophosphatidylinositol‐anchored glycoproteins and glycolipids) and glycan‐rich microvesicles that can fuse with the maturing sperm membrane. The sperm glycocalyx mediates numerous functions in the female reproductive tract, including the following: inhibition of premature capacitation; passage through the cervical mucus; protection from innate and adaptive female immunity; formation of the sperm reservoir; and masking sperm proteins involved in fertilization. The immense diversity in sperm‐associated glycans within and between species forms a remarkable challenge to our understanding of essential sperm glycan functions. Mol. Reprod. Dev. 82: 635–650, 2015. © 2015 The Authors. Molecular Reproduction and Development published by Wiley Periodicals, Inc. PMID:26061344

  15. Time-Dependent Measure of a Nano-Scale Force-Pulse Driven by the Axonemal Dynein Motors in Individual Live Sperm Cells

    SciTech Connect

    Allen, M J; Rudd, R E; McElfresh, M W; Balhorn, R

    2009-04-23

    Nano-scale mechanical forces generated by motor proteins are crucial to normal cellular and organismal functioning. The ability to measure and exploit such forces would be important to developing motile biomimetic nanodevices powered by biological motors for Nanomedicine. Axonemal dynein motors positioned inside the sperm flagellum drive microtubule sliding giving rise to rhythmic beating of the flagellum. This force-generating action makes it possible for the sperm cell to move through viscous media. Here we report new nano-scale information on how the propulsive force is generated by the sperm flagellum and how this force varies over time. Single cell recordings reveal discrete {approx}50 ms pulses oscillating with amplitude 9.8 {+-} 2.6 nN independent of pulse frequency (3.5-19.5 Hz). The average work carried out by each cell is 4.6 x 10{sup -16} J per pulse, equivalent to the hydrolysis of {approx}5,500 ATP molecules. The mechanochemical coupling at each active dynein head is {approx}2.2 pN/ATP, and {approx}3.9 pN per dynein arm, in agreement with previously published values obtained using different methods.

  16. Porcine embryo production following in vitro fertilization and intracytoplasmic sperm injection from vitrified immature oocytes matured with a granulosa cell co-culture system.

    PubMed

    Casillas, Fahiel; Ducolomb, Yvonne; Lemus, Ana E; Cuello, Cristina; Betancourt, Miguel

    2015-10-01

    This study was designed to evaluate the capacity of vitrified-warmed porcine immature oocytes to mature and to be fertilized using in vitro fertilization or intracytoplasmic sperm injection, and to determine the subsequent embryo development. Immature oocytes were vitrified using ethylene glycol and dimethylsulphoxide as cryoprotectants and the Cryolock method. After warming oocytes were cultured 44 h for maturation. Oocytes were randomly distributed in three treatment groups and subjected to in vitro fertilization (Experiment 1) or intracytoplasmic sperm injection (Experiment 2) procedures. The results indicate that the embryo development was higher in denuded oocytes co-cultured with granulosa cells (NkO-CC group) fertilized by in vitro fertilization or intracytoplasmic sperm injection compared to cumulus-cell oocyte complexes (COCs group), showing no significant differences with control. Vitrified denuded oocytes matured with a co-culture system NkO-CC group, displayed higher cleavage rate and blastocyst production than vitrified COCs group. Blastocysts were successfully obtained after IVF and ICSI procedures; however, the development to the blastocyst stage was better after IVF. These results show that the vitrification-warming media, the employment of a granulosa cell co-culture system and the Cryolock method during vitrification, increased the nuclear and cytoplasmic maturation of vitrified porcine immature oocytes. Further experiments are required to enhance porcine embryo production after vitrification.

  17. Accurate sperm morphology assessment predicts sperm function.

    PubMed

    Abu Hassan Abu, D; Franken, D R; Hoffman, B; Henkel, R

    2012-05-01

    Sperm morphology has been associated with in vitro as well as in vivo fertilisation. The study aimed to evaluate the possible relation between the percentage of spermatozoa with normal morphology and the following sperm functional assays: (i) zona-induced acrosome reaction (ZIAR); (ii) DNA integrity; (iii) chromatin condensation; (iv) sperm apoptosis; and (v) fertilisation rates. Regression analysis was employed to calculate the association between morphology and different functional tests. Normal sperm morphology correlated significantly with the percentages of live acrosome-reacted spermatozoa in the ZIAR (r = 0.518; P < 0.0001; n = 92), DNA integrity (r = -0.515; P = 0.0018; n = 34), CMA(3) -positive spermatozoa (r = -0.745; P < 0.0001; n = 92), sperm apoptosis (r = -0.395; P = 0.0206; n = 34) and necrosis (r = -0.545; P = 0.0009; n = 34). Negative correlations existed between for the acrosome reaction, and DNA integrity, while negative associations were recorded with the percentages of CMA(3) -positive spermatozoa, apoptotic and necrotic spermatozoa. Sperm morphology is related to sperm dysfunction such as poor chromatin condensation, acrosome reaction and DNA integrity. Negative and significant correlations existed between normal sperm morphology and chromatin condensation, the percentage of spermatozoa with abnormal DNA and spermatozoa with apoptotic activity. The authors do not regard sperm morphology as the only test for the diagnosis of male fertility, but sperm morphology can serve as a valuable indicator of underlying dysfunction.

  18. Identification of protein tyrosine phosphatases and dual-specificity phosphatases in mammalian spermatozoa and their role in sperm motility and protein tyrosine phosphorylation.

    PubMed

    González-Fernández, L; Ortega-Ferrusola, C; Macias-Garcia, B; Salido, G M; Peña, F J; Tapia, J A

    2009-06-01

    Protein tyrosine kinases have important roles in spermatozoa; however, little is known about the presence and regulation in these cells of their counterparts in signaling, namely, protein tyrosine phosphatases (PTPs) and dual-specificity phosphatases (DSPs). The objectives of the present study were to identify PTPs and DSPs in boar, stallion, and dog spermatozoa; to characterize their subcellular distribution; and to investigate the roles of tyrosine phosphatases in maintenance of protein tyrosine phosphorylation level and in sperm motility. Using Western blotting with specific antibodies in boar and stallion sperm lysates, we unequivocally identified two PTPs (PTPRB and PTPN11) and two DSPs (DUSP3 and DUSP4). In dog sperm lysates, only PTPN11, DUSP3, and DUSP4 were detected. In all these species, we did not detect the specific signal with anti-PTPRC (CD45), CDKN3, DUSP1, DUSP2, DUSP6, DUSP9, PTPN1, PTPN3, PTPN6, PTPN7, PTPN13, PTPRA, PTPRG, PTPRJ, PTPRK, or PTPRZ antibodies. Positive matches were further investigated by indirect immunofluorescence and confocal microscopy. Results showed that PTPRB was associated with the plasma membrane in the head and tail of boar and stallion spermatozoa. In agreement with Western blotting results, PTPRB antibodies did not show immunoreactivity in dog sperm analyzed by immunofluorescence. In the three species, DUSP4 was mainly found in the tail of spermatozoa, with little or no immunoreactivity in the head. PTPN11 was mainly located in the postacrosomal region in the head, whereas DUSP3 immunoreactivity was extended within the acrosome. PTPN11 and DUSP3 showed immunoreactivity in the tail that was restricted to the midpiece. Finally, we incubated boar, stallion, and dog spermatozoa with pervanadate and sodium orthovanadate, two PTP inhibitors, and analyzed overall protein tyrosine phosphorylation and assessed sperm motility. Sodium orthovanadate and pervanadate showed concentration-dependent inhibition of sperm motility that was

  19. Multitask learning for protein subcellular location prediction.

    PubMed

    Xu, Qian; Pan, Sinno Jialin; Xue, Hannah Hong; Yang, Qiang

    2011-01-01

    Protein subcellular localization is concerned with predicting the location of a protein within a cell using computational methods. The location information can indicate key functionalities of proteins. Thus, accurate prediction of subcellular localizations of proteins can help the prediction of protein functions and genome annotations, as well as the identification of drug targets. Machine learning methods such as Support Vector Machines (SVMs) have been used in the past for the problem of protein subcellular localization, but have been shown to suffer from a lack of annotated training data in each species under study. To overcome this data sparsity problem, we observe that because some of the organisms may be related to each other, there may be some commonalities across different organisms that can be discovered and used to help boost the data in each localization task. In this paper, we formulate protein subcellular localization problem as one of multitask learning across different organisms. We adapt and compare two specializations of the multitask learning algorithms on 20 different organisms. Our experimental results show that multitask learning performs much better than the traditional single-task methods. Among the different multitask learning methods, we found that the multitask kernels and supertype kernels under multitask learning that share parameters perform slightly better than multitask learning by sharing latent features. The most significant improvement in terms of localization accuracy is about 25 percent. We find that if the organisms are very different or are remotely related from a biological point of view, then jointly training the multiple models cannot lead to significant improvement. However, if they are closely related biologically, the multitask learning can do much better than individual learning.

  20. Mammalian sperm nuclear organization: resiliencies and vulnerabilities.

    PubMed

    Champroux, A; Torres-Carreira, J; Gharagozloo, P; Drevet, J R; Kocer, A

    2016-01-01

    Sperm cells are remarkably complex and highly specialized compared to somatic cells. Their function is to deliver to the oocyte the paternal genomic blueprint along with a pool of proteins and RNAs so a new generation can begin. Reproductive success, including optimal embryonic development and healthy offspring, greatly depends on the integrity of the sperm chromatin structure. It is now well documented that DNA damage in sperm is linked to reproductive failures both in natural and assisted conception (Assisted Reproductive Technologies [ART]). This manuscript reviews recent important findings concerning - the unusual organization of mammalian sperm chromatin and its impact on reproductive success when modified. This review is focused on sperm chromatin damage and their impact on embryonic development and transgenerational inheritance.

  1. Sperm competition differentially affects swimming velocity and size of spermatozoa from closely related muroid rodents: head first.

    PubMed

    Gómez Montoto, Laura; Varea Sánchez, María; Tourmente, Maximiliano; Martín-Coello, Juan; Luque-Larena, Juan José; Gomendio, Montserrat; Roldan, Eduardo R S

    2011-12-01

    Sperm competition favours an increase in sperm swimming velocity that maximises the chances that sperm will reach the ova before rival sperm and fertilise. Comparative studies have shown that the increase in sperm swimming speed is associated with an increase in total sperm size. However, it is not known which are the first evolutionary steps that lead to increases in sperm swimming velocity. Using a group of closely related muroid rodents that differ in levels of sperm competition, we here test the hypothesis that subtle changes in sperm design may represent early evolutionary changes that could make sperm swim faster. Our findings show that as sperm competition increases so does sperm swimming speed. Sperm swimming velocity is associated with the size of all sperm components. However, levels of sperm competition are only related to an increase in sperm head area. Such increase is a consequence of an increase in the length of the sperm head, and also of the presence of an apical hook in some of the species studied. These findings suggest that the presence of a hook may modify the sperm head in such a way that would help sperm swim faster and may also be advantageous if sperm with larger heads are better able to attach to the epithelial cells lining the lower isthmus of the oviduct where sperm remain quiescent before the final race to reach the site of fertilisation.

  2. Identification of endogenous metabolites in human sperm cells using proton nuclear magnetic resonance ((1) H-NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS).

    PubMed

    Paiva, C; Amaral, A; Rodriguez, M; Canyellas, N; Correig, X; Ballescà, J L; Ramalho-Santos, J; Oliva, R

    2015-05-01

    The objective of this study was to contribute to the first comprehensive metabolomic characterization of the human sperm cell through the application of two untargeted platforms based on proton nuclear magnetic resonance ((1) H-NMR) spectroscopy and gas chromatography coupled to mass spectrometry (GC-MS). Using these two complementary strategies, we were able to identify a total of 69 metabolites, of which 42 were identified using NMR, 27 using GC-MS and 4 by both techniques. The identity of some of these metabolites was further confirmed by two-dimensional (1) H-(1) H homonuclear correlation spectroscopy (COSY) and (1) H-(13) C heteronuclear single-quantum correlation (HSQC) spectroscopy. Most of the metabolites identified are reported here for the first time in mature human spermatozoa. The relationship between the metabolites identified and the previously reported sperm proteome was also explored. Interestingly, overrepresented pathways included not only the metabolism of carbohydrates, but also of lipids and lipoproteins. Of note, a large number of the metabolites identified belonged to the amino acids, peptides and analogues super class. The identification of this initial set of metabolites represents an important first step to further study their function in male gamete physiology and to explore potential reasons for dysfunction in future studies. We also demonstrate that the application of NMR and MS provides complementary results, thus constituting a promising strategy towards the completion of the human sperm cell metabolome.

  3. Development of a lipopolysaccharide (LPS)-supplemented adjuvant and its effects on cell-mediated and humoral immune responses in male rats immunized against sperm

    PubMed Central

    NOGUCHI, Junko; WATANABE, Shinya; NGUYEN, Thanh Q. Dang; KIKUCHI, Kazuhiro; KANEKO, Hiroyuki

    2016-01-01

    Supplementation with lipopolysaccharide (LPS) from non-pathogenic Escherichia coli was found to enhance the adjuvant effects of a veterinary vaccine adjuvant (ISA 71VG®). Sperm immunization using 71VG as an adjuvant in the immature period induced infertility in 25% of male rats, whereas this increased to 62.5% after immunization with 71VG + LPS or Freund′s complete adjuvant (FCA). Mean testicular weight of non-sterile males in the 71VG + LPS group was significantly lower than that in the 71VG or FCA group. Histological examination of testicular tissue from sterile males demonstrated severe impairment of spermatogenesis due to experimental autoimmune orchitis, a cell-mediated autoimmune condition. The serum anti-sperm titer was elevated in the three sperm-immunized groups relative to male rats treated with adjuvant alone, but the titer was higher in the 71VG + LPS and FCA groups than in the 71VG group. We consider that this LPS-supplemented adjuvant stimulates both humoral and cell-mediated immune responses to an extent comparable to FCA. PMID:27890874

  4. Delivery following intracytoplasmic injection of mature sperm cells recovered by testicular fine needle aspiration in a case of hypergonadotropic azoospermia due to maturation arrest.

    PubMed

    Lewin, A; Weiss, D B; Friedler, S; Ben-Shachar, I; Porat-Katz, A; Meirow, D; Schenker, J G; Safran, A

    1996-04-01

    This is the first reported delivery following intracytoplasmic sperm injection (ICSI) of mature live testicular sperm cells collected in a case of hypergonadotrophic azoospermia with maturation arrest. The 30 year old couple presented with primary infertility of 11 years duration, the man being submitted in childhood to five orchidopexy operations for the treatment of cryptorchism. He had elevated serum follicle stimulating hormone (FSH; 18.8 IU/I), an atrophic left testis and a normal sized right testis, the biopsy of which diagnosed maturation arrest and focal scarring. The couple refused donor insemination for religious reasons and the only option was an attempt at testicular sperm collection. Multiple testicular and epididymal fine needle aspirations were performed, using an aspiration handle loaded with 20 ml syringe and 21-23 gauge butterfly needles. The mature spermatozoa recovered were used to inseminate the oocytes by ICSI. Prior to this procedure, the patient's wife underwent ovulation induction using a long protocol of mid-luteal gonadotrophin-releasing hormone analogue/human menopausal gonadotrophin (GnRHa/HMG). At oocyte retrieval, ten oocytes were recovered. Eight live sperm cells were recovered from the aspirates of the right testis. Following ICSI into four metaphase II and two metaphase I oocytes, one mature oocyte was fertilized, cleaved and was transferred to the uterus 48 h after oocyte retrieval. The patient conceived and delivered a 3300 g boy at term. In conclusion, our results demonstrate that this novel approach should be considered in cases with hypergonadotrophic azoospermia due to testicular failure. Further experience is needed to establish the exact criteria for its use.

  5. Geometric Morphometrics of Rodent Sperm Head Shape

    PubMed Central

    Varea Sánchez, María; Bastir, Markus; Roldan, Eduardo R. S.

    2013-01-01

    Mammalian spermatozoa, particularly those of rodent species, are extremely complex cells and differ greatly in form and dimensions. Thus, characterization of sperm size and, particularly, sperm shape represents a major challenge. No consensus exists on a method to objectively assess size and shape of spermatozoa. In this study we apply the principles of geometric morphometrics to analyze rodent sperm head morphology and compare them with two traditional morphometry methods, that is, measurements of linear dimensions and dimensions-derived parameters calculated using formulae employed in sperm morphometry assessments. Our results show that geometric morphometrics clearly identifies shape differences among rodent spermatozoa. It is also capable of discriminating between size and shape and to analyze these two variables separately. Thus, it provides an accurate method to assess sperm head shape. Furthermore, it can identify which sperm morphology traits differ between species, such as the protrusion or retraction of the base of the head, the orientation and relative position of the site of flagellum insertion, the degree of curvature of the hook, and other distinct anatomical features and appendices. We envisage that the use of geometric morphometrics may have a major impact on future studies focused on the characterization of sperm head formation, diversity of sperm head shape among species (and underlying evolutionary forces), the effects of reprotoxicants on changes in cell shape, and phenotyping of genetically-modified individuals. PMID:24312234

  6. SIMS ion microscopy imaging of boronophenylalanine (BPA) and 13C15N-labeled phenylalanine in human glioblastoma cells: Relevance of subcellular scale observations to BPA-mediated boron neutron capture therapy of cancer

    NASA Astrophysics Data System (ADS)

    Chandra, Subhash; Lorey, Daniel R., II

    2007-02-01

    p-Boronophenylalanine (BPA) is a clinically approved boron neutron capture therapy (BNCT) agent currently being used in clinical trials of glioblastoma multiforme, melanoma and liver metastases. Secondary ion mass spectrometry (SIMS) observations from the Cornell SIMS Laboratory provided support for using a 6 h infusion of BPA, instead of a 2 h infusion, for achieving higher levels of boron in brain tumor cells. These observations were clinically implemented in Phase II experimental trials of glioblastoma multiforme in Sweden. However, the mechanisms for higher BPA accumulation with longer infusions have remained unknown. In this work, by using 13C15N-labeled phenylalanine and T98G human glioblastoma cells, comparisons between the 10B-delivery of BPA and the accumulation of labeled phenylalanine after 2 and 6 h treatments were made with a Cameca IMS-3f SIMS ion microscope at 500 nm spatial resolution in fast frozen, freeze-fractured, freeze-dried cells. Due to the presence of the Na-K-ATPase in the plasma membrane of most mammalian cells, the cells maintain an approximately 10/1 ratio of K/Na in the intracellular milieu. Therefore, the quantitative imaging of these highly diffusible species in the identical cell in which the boron or labeled amino acid was imaged provides a rule-of-thumb criterion for validation of SIMS observations and the reliability of the cryogenic sampling. The labeled phenylalanine was detected at mass 28, as the 28(13C15N)- molecular ion. Correlative analysis with optical and confocal laser scanning microscopy revealed that fractured freeze-dried glioblastoma cells contained well-preserved ultrastructural details with three discernible subcellular regions: a nucleus or multiple nuclei, a mitochondria-rich perinuclear cytoplasmic region and the remaining cytoplasm. SIMS analysis revealed that the overall cellular signals of both 10B from BPA and 28CN- from labeled phenylalanine increased approximately 1.6-fold between the 2 and 6 h exposures

  7. Subcellular compartmentation of glutathione in dicotyledonous plants

    PubMed Central

    Müller, Maria

    2010-01-01

    This study describes the subcellular distribution of glutathione in roots and leaves of different plant species (Arabidopsis, Cucurbita, and Nicotiana). Glutathione is an important antioxidant and redox buffer which is involved in many metabolic processes including plant defense. Thus information on the subcellular distribution in these model plants especially during stress situations provides a deeper insight into compartment specific defense reactions and reflects the occurrence of compartment specific oxidative stress. With immunogold cytochemistry and computer-supported transmission electron microscopy glutathione could be localized in highest contents in mitochondria, followed by nuclei, peroxisomes, the cytosol, and plastids. Within chloroplasts and mitochondria, glutathione was restricted to the stroma and matrix, respectively, and did not occur in the lumen of cristae and thylakoids. Glutathione was also found at the membrane and in the lumen of the endoplasmic reticulum. It was also associated with the trans and cis side of dictyosomes. None or only very little glutathione was detected in vacuoles and the apoplast of mesophyll and root cells. Additionally, glutathione was found in all cell compartments of phloem vessels, vascular parenchyma cells (including vacuoles) but was absent in xylem vessels. The specificity of this method was supported by the reduction of glutathione labeling in all cell compartments (up to 98%) of the glutathione-deficient Arabidopsis thaliana rml1 mutant. Additionally, we found a similar distribution of glutathione in samples after conventional fixation and rapid microwave-supported fixation. Thus, indicating that a redistribution of glutathione does not occur during sample preparation. Summing up, this study gives a detailed insight into the subcellular distribution of glutathione in plants and presents solid evidence for the accuracy and specificity of the applied method. PMID:20186447

  8. GAR22β regulates cell migration, sperm motility, and axoneme structure

    PubMed Central

    Gamper, Ivonne; Fleck, David; Barlin, Meltem; Spehr, Marc; Sayad, Sara El; Kleine, Henning; Maxeiner, Sebastian; Schalla, Carmen; Aydin, Gülcan; Hoss, Mareike; Litchfield, David W.; Lüscher, Bernhard; Zenke, Martin; Sechi, Antonio

    2016-01-01

    Spatiotemporal cytoskeleton remodeling is pivotal for cell adhesion and migration. Here we investigated the function of Gas2-related protein on chromosome 22 (GAR22β), a poorly characterized protein that interacts with actin and microtubules. Primary and immortalized GAR22β−/− Sertoli cells moved faster than wild-type cells. In addition, GAR22β−/− cells showed a more prominent focal adhesion turnover. GAR22β overexpression or its reexpression in GAR22β−/− cells reduced cell motility and focal adhesion turnover. GAR22β–actin interaction was stronger than GAR22β–microtubule interaction, resulting in GAR22β localization and dynamics that mirrored those of the actin cytoskeleton. Mechanistically, GAR22β interacted with the regulator of microtubule dynamics end-binding protein 1 (EB1) via a novel noncanonical amino acid sequence, and this GAR22β–EB1 interaction was required for the ability of GAR22β to modulate cell motility. We found that GAR22β is highly expressed in mouse testes, and its absence resulted in reduced spermatozoa generation, lower actin levels in testes, and impaired motility and ultrastructural disorganization of spermatozoa. Collectively our findings identify GAR22β as a novel regulator of cell adhesion and migration and provide a foundation for understanding the molecular basis of diverse cytoskeleton-dependent processes. PMID:26564797

  9. Importance of sperm morphology during sperm transport and fertilization in mammals

    PubMed Central

    García-Vázquez, Francisco A; Gadea, Joaquín; Matás, Carmen; Holt, William V

    2016-01-01

    After natural or artificial insemination, the spermatozoon starts a journey from the site of deposition to the place of fertilization. However, only a small subset of the spermatozoa deposited achieves their goal: to reach and fertilize the egg. Factors involved in controlling sperm transport and fertilization include the female reproductive tract environment, cell-cell interactions, gene expression, and phenotypic sperm traits. Some of the significant determinants of fertilization are known (i.e., motility or DNA status), but many sperm traits are still indecipherable. One example is the influence of sperm dimensions and shape upon transport within the female genital tract towards the oocyte. Biophysical associations between sperm size and motility may influence the progression of spermatozoa through the female reproductive tract, but uncertainties remain concerning how sperm morphology influences the fertilization process, and whether only the sperm dimensions per se are involved. Moreover, such explanations do not allow the possibility that the female tract is capable of distinguishing fertile spermatozoa on the basis of their morphology, as seems to be the case with biochemical, molecular, and genetic properties. This review focuses on the influence of sperm size and shape in evolution and their putative role in sperm transport and selection within the uterus and the ability to fertilize the oocyte. PMID:27624988

  10. Tolerance to environmental desiccation in moss sperm.

    PubMed

    Shortlidge, Erin E; Rosenstiel, Todd N; Eppley, Sarah M

    2012-05-01

    • Sexual reproduction in mosses requires that sperm be released freely into the environment before finding and fertilizing a receptive female. After release from the male plant, moss sperm may experience a range of abiotic stresses; however, few data are available examining stress tolerance of moss sperm and whether there is genetic variation for stress tolerance in this important life stage. • Here, we investigated the effects of environmental desiccation and recovery on the sperm cells of three moss species (Bryum argenteum, Campylopus introflexus, and Ceratodon purpureus). • We found that a fraction of sperm cells were tolerant to environmental desiccation for extended periods (d) and that tolerance did not vary among species. We found that this tolerance occurs irrespective of ambient dehydration conditions, and that the addition of sucrose during dry-down improved cell recovery. Although we observed no interspecific variation, significant variation among individuals within species in sperm cell tolerance to environmental desiccation was observed, suggesting selection could potentially act on this basic reproductive trait. • The observation of desiccation-tolerant sperm in multiple moss species has important implications for understanding bryophyte reproduction, suggesting the presence of a significant, uncharacterized complexity in the ecology of moss mating systems.

  11. Magnetic activated cell sorting: an effective method for reduction of sperm DNA fragmentation in varicocele men prior to assisted reproductive techniques.

    PubMed

    Degheidy, T; Abdelfattah, H; Seif, A; Albuz, F K; Gazi, S; Abbas, S

    2015-10-01

    Semen parameters of varicocele men have been usually suspected to exhibit higher levels of abnormalities including DNA fragmentation, reactive oxygen species (ROS) and apoptotic markers. Negative correlation between increased level of DNA fragmentation and assisted reproductive techniques (ART) outcome has been studied by several authors. In the current study, we aim to evaluate the possible value of magnetic activated cell sorting (MACs) technology in reduction of DNA fragmentation in infertile varicocele patients prior to ART. Semen samples, collected from 36 varicocele patients, were prepared by density gradient centrifugation (DGC). Every sample was subsequently divided into two aliquots. One aliquot was kept untouched as pre-MACs control while the other aliquot was subjected to MACs technique, for depletion of apoptotic spermatozoa, and serves as post-MACs test. Sperm count, motility and DNA fragmentations were evaluated for both control and test samples. Post-MACs samples showed no deleterious reduction in total sperm motility (80.64 ± 6.97%) compared with control samples (80.97 ± 7.74%) while sperm DNA fragmentations were significantly reduced in post-MACs samples (9.61 ± 5.62%) compared with pre-MACs controls (12.43 ± 6.29%) (P < 0.05). It can be concluded that MACs technique is a simple, noninvasive, technique that can efficiently reduce DNA fragmentation in infertile varicocele patients prior to ART.

  12. Liver X receptor ligand cytotoxicity in colon cancer cells and not in normal colon epithelial cells depends on LXRβ subcellular localization.

    PubMed

    Courtaut, Flavie; Derangère, Valentin; Chevriaux, Angélique; Ladoire, Sylvain; Cotte, Alexia K; Arnould, Laurent; Boidot, Romain; Rialland, Mickaël; Ghiringhelli, François; Rébé, Cédric

    2015-09-29

    Increasing evidence indicates that Liver X Receptors (LXRs) have some anticancer properties. We recently demonstrated that LXR ligands induce colon cancer cell pyroptosis through an LXRβ-dependent pathway. In the present study, we showed that human colon cancer cell lines presented differential cytoplasmic localizations of LXRβ. This localization correlated with caspase-1 activation and cell death induction under treatment with LXR ligand. The association of LXRβ with the truncated form of RXRα (t-RXRα) was responsible for the sequestration of LXRβ in the cytoplasm in colon cancer cells. Moreover t-RXRα was not expressed in normal colon epithelial cells. These cells presented a predominantly nuclear localization of LXRβ and were resistant to LXR ligand cytotoxicity. Our results showed that predominant cytoplasmic localization of LXRβ, which occurs in colon cancer cells but not in normal colon epithelial cells, allowed LXR ligand-induced pyroptosis. This study strengthens the hypothesis that LXRβ could be a promising target in cancer therapy.

  13. Induction of micronuclei and sister chromatid exchange in bone-marrow cells and abnormalities in sperm of Algerian mice (Mus spretus) exposed to cadmium, lead and zinc.

    PubMed

    Tapisso, Joaquim Torres; Marques, Carla Cristina; Mathias, Maria da Luz; Ramalhinho, Maria da Graça

    2009-08-01

    As a consequence of human activities, large amounts of cadmium, lead and zinc are released in the environment, often simultaneously. The aim of this study was to investigate under experimental conditions the DNA damage induced in Algerian mice (Mus spretus) exposed to cadmium (Cd), lead (Pb) and zinc (Zn) separately, or in selected combinations. Three cytogenetic end points were considered: the frequencies of micronucleated cells (MN) and sister chromatid exchange (SCE) in the bone marrow and the frequency of sperm abnormalities. Mice were treated by intraperitoneal (i.p.) injections with 5 or 10 doses of aqueous solutions of cadmium acetate, lead acetate and zinc acetate in concentrations corresponding to 1/10 of the LD50, respectively, 21.5, 0.46 and 1.5 mg/kg bw. The control groups were injected in the same way with distilled water. With only one exception (Cd + Zn group treated with 5 doses), the results show a significant increase of MN in all groups for both treatments (5 and 10 doses). Similarly, the results concerning the SCE revealed a statistically significant increase in all treated animals, with the exception of the Zn group treated with 5 doses. The number of sperm abnormalities was significantly higher in animals treated with 5 doses, except in the group Pb + Zn. In animals treated with 10 doses the number of sperm abnormalities was always statistically higher compared with controls. This study indicates that cadmium, lead and zinc can induce MN, SCEs and sperm abnormalities in Algerian mice and that the clastogenic potential is dependent on the time of exposure and the interaction between the three elements, confirming the environmental damage that may result from the simultaneous action of several metals. Most relevant is the toxic potential for Zn, related with the dose, which may compromise its protective effect against other metal contaminations, such as cadmium.

  14. Direct binding of boar ejaculate and epididymal spermatozoa to porcine epididymal epithelial cells is also needed to maintain sperm survival in in vitro co-culture.

    PubMed

    Yeste, Marc; Castillo-Martín, Míriam; Bonet, Sergi; Briz, Maria Dolors

    2012-04-01

    The aim of the present study was to compare the influence of cultured epididymal epithelial cells (EEC) from corpus, caput or cauda, oviductal epithelial cells (OEC) and non-reproductive epithelial cells (LLC-PK1) on function and survival of epididymal and ejaculated spermatozoa, in the latter case to determine whether such influence differed between morphologically normal and abnormal spermatozoa. For this purpose, either spermatozoa were directly co-cultured with EEC from caput, corpus, or cauda, OEC and LLC-PK1 cells (experiment 1) or a membrane-diffusible insert was included in these co-cultures (experiment 2). EEC cultured from the three epididymal regions did not differently affect the sperm parameters. Morphologically normal spermatozoa presented a higher ability to bind EEC, OEC, and LLC-PK1 than abnormal spermatozoa with cytoplasmic droplets or with tail/head malformations. Epididymal spermatozoa were more able to bind EEC during the first 24 h of co-culture, while ejaculated spermatozoa presented a higher capacity to bind OEC between 30 min and 3 h of co-incubation. In all cases, the ability to bind to epithelial cells was higher when they were co-cultured with EEC and OEC than with LLC-PK1. After 2 h of co-culture, the viability of epididymal spermatozoa was better maintained when they bound EEC than when they bound OEC. Conversely, the viability of ejaculated spermatozoa was better maintained when bound OEC than when bound EEC after 24 and 48 h of co-culture. Our work, apart from corroborating the involvement of morphologically normal spermatozoa in the formation of sperm reservoir, highlights the importance of direct contact spermatozoa-EEC in maintaining the sperm survival in in vitro co-culture, and also suggests that a specific binding between EEC and epididymal spermatozoa exists.

  15. [The contractile heart. Cellular and subcellular aspects].

    PubMed

    Larsen, T H

    1994-12-10

    The excitation-contraction coupling can be defined as the mechanisms involved when the action potential initiates a contraction response of the cardiac muscle cell. The action potential is conducted from cell to cell, resulting in a synchronized contraction of the myocardium. Both the propagation of the action potential and the myofibrillar contraction are dependent on changes in free Ca++ concentrations. Recently, the mediators and the molecular and structural components involved in the subcellular transforming of the action potential into a contraction have been characterized. The opening of voltage-dependent L-type Ca(++)-channels in the cell membrane stimulates a release of Ca++ from the sarcoplasmic reticulum. This Ca(++)-mediated Ca(++)-release appears to be a graded mechanism and is associated with the presence of structural couplings (Ca++ synapses) in the cardiac muscle cell.

  16. Flow cytometry of sperm

    SciTech Connect

    Gledhill, B.L.

    1987-09-21

    This brief paper summarizes automated flow cytometric determination of sperm morphology and flow cytometry/sorting of sperm with application to sex preselection. In the latter context, mention is made of results of karyotypic determination of sex chromosome ratios in albumin-processed human sperm. 23 refs., 1 fig., 1 tab.

  17. Chemical dispersants used in the Gulf of Mexico oil crisis are cytotoxic and genotoxic to sperm whale skin cells.

    PubMed

    Wise, Catherine F; Wise, James T F; Wise, Sandra S; Thompson, W Douglas; Wise, John Pierce; Wise, John Pierce

    2014-07-01

    The 2010 Deepwater Horizon oil rig explosion in the Gulf of Mexico drew attention to the need for toxicological studies of chemical dispersants. We are still learning the effects these spills had on wildlife. Little is known about the toxicity of these substances in marine mammals. The objective of this study was to determine the toxicity of the two dispersants (Corexit 9500 and 9527). Corexit 9500 and 9527 were both cytotoxic to sperm whale skin fibroblasts. Corexit 9527 was less cytotoxic than 9500. S9 mediated metabolism did not alter cytotoxicity of either dispersant. Both dispersants were genotoxic to sperm whale skin fibroblasts; S9 mediated metabolism increased Corexit 9527 genotoxicity.

  18. Cytogenetics of human sperm: Structural aberrations and DNA replication

    SciTech Connect

    Brandriff, B.F.; Gordon, L.A.; Carrano, A.V.

    1989-07-11

    The human sperm-hamster egg system, first introduced in 1978 (Rudak et al), has yielded some important insights into questions on chromosomal integrity of human sperm. In this system, human sperm are co-incubated with eggs from the golden hamster. After the gametes fuse, eggs are cultured overnight and approximately 15 hours after fusion, display the haploid chromosomal complement of individual human sperm cells. These chromosomes can be analyzed by standard banding techniques to identify and quantify structural and numerical abnormalities in single sperm. 32 refs., 1 fig.

  19. Intracellular pH (pHin) and cytosolic calcium ([Ca2+]cyt) regulation via ATPases: studies in cell populations, single cells, and subcellular compartments

    NASA Astrophysics Data System (ADS)

    Rojas, Jose D.; Sanka, Shankar C.; Gyorke, Sandor; Wesson, Donald E.; Minta, Akwasi; Martinez-Zaguilan, Raul

    1999-07-01

    Changes in pHin and (Ca2+)cyt are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca2+ regulatory mechanism might be altered. To study these ions, we employ fluorescence spectroscopy, and cell imagin spectroscopy/confocal microscopy. pH and Ca2+ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (E/L) compartments by overnight incubation of cells with dextran- conjugated ion fluorescent probes. We focus on specific pH and Ca2+ regulatory systems: plasmalemmal vacuolar- type H+-ATPases (pm V-ATPases) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). As experimental models, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca2+ homeostasis in both VSM and microvascular endothelial cells; (b) E/L compartments are important for pH and Ca2+ regulation via H+-ATPases and SERCA, respectively; and (c) pm-V- ATPases are important for pHin regulation in microvascular endothelial cells.

  20. Lactic acid is a sperm motility inactivation factor in the sperm storage tubules

    PubMed Central

    Matsuzaki, Mei; Mizushima, Shusei; Hiyama, Gen; Hirohashi, Noritaka; Shiba, Kogiku; Inaba, Kazuo; Suzuki, Tomohiro; Dohra, Hideo; Ohnishi, Toshiyuki; Sato, Yoshikatsu; Kohsaka, Tetsuya; Ichikawa, Yoshinobu; Atsumi, Yusuke; Yoshimura, Takashi; Sasanami, Tomohiro

    2015-01-01

    Although successful fertilization depends on timely encounters between sperm and egg, the decoupling of mating and fertilization often confers reproductive advantages to internally fertilizing animals. In several vertebrate groups, postcopulatory sperm viability is prolonged by storage in specialized organs within the female reproductive tract. In birds, ejaculated sperm can be stored in a quiescent state within oviductal sperm storage tubules (SSTs), thereby retaining fertilizability for up to 15 weeks at body temperature (41 °C); however, the mechanism by which motile sperm become quiescent within SSTs is unknown. Here, we show that low oxygen and high lactic acid concentrations are established in quail SSTs. Flagellar quiescence was induced by lactic acid in the concentration range found in SSTs through flagellar dynein ATPase inactivation following cytoplasmic acidification (sperm morphology under hypoxic and high temperature conditions indicates that a combination of these factors enables sperm cells to survive during the ovulation cycles. Our findings suggested a novel physiological role for lactic acid in promoting sperm quiescence in SSTs and opened up a new opportunity for technological improvement in prolonging sperm longevity at ambient or body temperature. PMID:26619826

  1. Protein subcellular localization assays using split fluorescent proteins

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2009-09-08

    The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

  2. Ca2+/Calmodulin-Dependent Protein Kinase Kinases (CaMKKs) Effects on AMP-Activated Protein Kinase (AMPK) Regulation of Chicken Sperm Functions

    PubMed Central

    Nguyen, Thi Mong Diep; Combarnous, Yves; Praud, Christophe; Duittoz, Anne; Blesbois, Elisabeth

    2016-01-01

    Sperm require high levels of energy to ensure motility and acrosome reaction (AR) accomplishment. The AMP-activated protein kinase (AMPK) has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca2+/calmodulin-dependent protein kinase kinases (CaMKKs) mediated pathway. The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca2+, or of CaMKKs inhibitor (STO-609). Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β), CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca2+ but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca2+ than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca2+. Our results show for the first time the presence of CaMKKs (α and β) and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca2+ entry in sperm through the Ca2+/CaM/CaMKKs/CaMKI pathway. The Ca2+/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca2+ entry in the cells

  3. Differences in the fatty-acid composition of rodent spermatozoa are associated to levels of sperm competition.

    PubMed

    delBarco-Trillo, Javier; Mateo, Rafael; Roldan, Eduardo R S

    2015-03-20

    Sperm competition is a prevalent phenomenon that drives the evolution of sperm function. High levels of sperm competition lead to increased metabolism to fuel higher sperm velocities. This enhanced metabolism can result in oxidative damage (including lipid peroxidation) and damage to the membrane. We hypothesized that in those species experiencing high levels of sperm competition there are changes in the fatty-acid composition of the sperm membrane that makes the membrane more resistant to oxidative damage. Given that polyunsaturated fatty acids (PUFAs) are the most prone to lipid peroxidation, we predicted that higher sperm competition leads to a reduction in the proportion of sperm PUFAs. In contrast, we predicted that levels of sperm competition should not affect the proportion of PUFAs in somatic cells. To test these predictions, we quantified the fatty-acid composition of sperm, testis and liver cells in four mouse species (genus Mus) that differ in their levels of sperm competition. Fatty-acid composition in testis and liver cells was not associated to sperm competition levels. However, in sperm cells, as predicted, an increase in sperm competition levels was associated with an increase in the proportion of saturated fatty-acids (the most resistant to lipid peroxidation) and by a concomitant decrease in the proportion of PUFAs. Two particular fatty acids were most responsible for this pattern (arachidonic acid and palmitic acid). Our findings thus indicate that sperm competition has a pervasive influence in the composition of sperm cells that ultimately may have important effects in sperm function.

  4. Etiologies of sperm oxidative stress

    PubMed Central

    Sabeti, Parvin; Pourmasumi, Soheila; Rahiminia, Tahereh; Akyash, Fatemeh; Talebi, Ali Reza

    2016-01-01

    Sperm is particularly susceptible to reactive oxygen species (ROS) during critical phases of spermiogenesis. However, the level of seminal ROS is restricted by seminal antioxidants which have beneficial effects on sperm parameters and developmental potentials. Mitochondria and sperm plasma membrane are two major sites of ROS generation in sperm cells. Besides, leukocytes including polymer phonuclear (PMN) leukocytes and macrophages produce broad category of molecules including oxygen free radicals, non-radical species and reactive nitrogen species. Physiological role of ROS increase the intracellular cAMP which then activate protein kinase in male reproductive system. This indicates that spermatozoa need small amounts of ROS to acquire the ability of nuclear maturation regulation and condensation to fertilize the oocyte. There is a long list of intrinsic and extrinsic factors which can induce oxidative stress to interact with lipids, proteins and DNA molecules. As a result, we have lipid peroxidation, DNA fragmentation, axonemal damage, denaturation of the enzymes, over generation of superoxide in the mitochondria, lower antioxidant activity and finally abnormal spermatogenesis. If oxidative stress is considered as one of the main cause of DNA damage in the germ cells, then there should be good reason for antioxidant therapy in these conditions. PMID:27351024

  5. Magnetic hyperthermia and pH-responsive effective drug delivery to the sub-cellular level of human breast cancer cells by modified CoFe2O4 nanoparticles.

    PubMed

    Oh, Yunok; Moorthy, Madhappan Santha; Manivasagan, Panchanathan; Bharathiraja, Subramaniyan; Oh, Junghwan

    2017-02-01

    Magnetic iron oxide nanoparticles (MNPs) have been extensively utilized in a wide range of biomedical applications including magnetic hyperthermia agent. To improve the efficiency of the MNPs in therapeutic applications, in this study, we have synthesized CoFe2O4 nanoparticles and its surface was further functionalized with meso-2,3-dimercaptosuccinic acid (DMSA). The anticancer agent, Doxorubucin (DOX) was conjugated with CoFe2O4@DMSA nanoparticle to evaluate the combined effects of thermotherapy and chemotherapy. The drug delivery efficiency of the DOX loaded CoFe2O4@DMSA nanoparticles were examined based on magnetically triggered delivery of DOX into the subcellular level of cancer cells by using MDA-MB-231 cell line. The amine part of the DOX molecules were effectively attached through an electrostatic interactions and/or hydrogen bonding interactions with the carboxylic acid groups of the DMSA functionalities present onto the surface of the CoFe2O4 nanoparticles. The DOX loaded CoFe2O4@DMSA nanoparticles can effectively uptake with cancer cells via typical endocytosis process. After endocytosis, DOX release from CoFe2O4 nanoparticles was triggered by intracellular endosomal/lysosomal acidic environments and the localized heat can be generated under an alternating magnetic field (AMF). In the presence of AMF, the released DOX molecules were accumulated with high concentrations into the subcellular level at a desired sites and exhibited a synergistic effect of an enhanced cell cytotoxicity by the combined effects of thermal-chemotherapy. Importantly, pH- and thermal-responsive Dox-loaded CoFe2O4 nanoparticles induced significant cellular apoptosis more efficiently mediated by active mitochondrial membrane and ROS generation than the free Dox. Thus, the Dox-loaded CoFe2O4@DMSA nanoparticles can be used as a potential therapeutic agent in cancer therapy by combining the thermo-chemotherapy techniques.

  6. Paternal exposure to testis cancer chemotherapeutics alters sperm fertilizing capacity and affects gene expression in the eight-cell stage rat embryo.

    PubMed

    Maselli, J; Hales, B F; Robaire, B

    2014-03-01

    Treatment of testicular cancer includes the coadministration of bleomycin, etoposide and cis-platinum (BEP); however, along with its therapeutic benefit, BEP exposure results in extensive reproductive chemotoxic effects, including alterations to sperm chromatin integrity. As an intact paternal genome is essential for successful fertilization and embryogenesis, we assessed the effect of paternal exposure to BEP on sperm fertilization capacity and the resulting consequences on early embryonic gene expression. Adult male Brown Norway rats received a 9-week treatment with BEP or saline and then were sacrificed immediately or subject to a 9-week recovery period. HSP90AA1, HSP90B1 and PDIA3, involved in spermatozoa-egg interactions, were overexpressed in BEP-exposed spermatozoa after the 9-week treatment period; overexpression was also observed in spermatozoa from BEP-treated rats after 9 weeks of recovery. These proteins were localized to the plasma membrane of the sperm head; this localization may facilitate their role in spermatozoa-egg interactions as the highest staining intensities were observed in capacitated spermatozoa. The fertilization potential of spermatozoa was determined by in vitro fertilization with oocytes from unexposed naturally cycling female rats. Interestingly, the fertilization potential of spermatozoa following a 9-week recovery period from BEP treatment was significantly enhanced compared with controls. Moreover, stem cell transcription factors, involved in the regulation of a plethora of early embryonic events, were upregulated by more than twofold in eight-cell stage embryos sired by BEP recovery males compared with controls; this suggests that there are potential deleterious effects on embryo development well after termination of BEP exposure.

  7. Not All Sperm Are Equal: Functional Mitochondria Characterize a Subpopulation of Human Sperm with Better Fertilization Potential

    PubMed Central

    Sousa, Ana Paula; Amaral, Alexandra; Baptista, Marta; Tavares, Renata; Caballero Campo, Pedro; Caballero Peregrín, Pedro; Freitas, Albertina; Paiva, Artur; Almeida-Santos, Teresa; Ramalho-Santos, João

    2011-01-01

    Human sperm samples are very heterogeneous and include a low amount of truly functional gametes. Distinct strategies have been developed to characterize and isolate this specific subpopulation. In this study we have used fluorescence microscopy and fluorescence-activated cell sorting to determine if mitochondrial function, as assessed using mitochondrial-sensitive probes, could be employed as a criterion to obtain more functional sperm from a given ejaculate. We first determined that mitochondrial activity correlated with the quality of distinct human samples, from healthy donors to patients with decreased semen quality. Furthermore, using fluorescence-activated cell sorting to separate sperm with active and inactive mitochondria we found that this was also true within samples. Indeed, sperm with active mitochondria defined a more functional subpopulation, which contained more capacitated and acrosome intact cells, sperm with lower chromatin damage, and, crucially, sperm more able to decondense and participate in early development using both chemical induction and injection into mature bovine oocytes. Furthermore, cell sorting using mitochondrial activity produced a more functional sperm subpopulation than classic swim-up, both in terms of improvement in a variety of functional sperm parameters and in statistical significance. In conclusion, whatever the true biological role of sperm mitochondria in fertilization, mitochondrial activity is a clear hallmark of human sperm functionality. PMID:21448461

  8. The dynamics of sperm cooperation in a competitive environment

    PubMed Central

    Fisher, Heidi S.; Giomi, Luca; Hoekstra, Hopi E.; Mahadevan, L.

    2014-01-01

    Sperm cooperation has evolved in a variety of taxa and is often considered a response to sperm competition, yet the benefit of this form of collective movement remains unclear. Here, we use fine-scale imaging and a minimal mathematical model to study sperm aggregation in the rodent genus Peromyscus. We demonstrate that as the number of sperm cells in an aggregate increase, the group moves with more persistent linearity but without increasing speed. This benefit, however, is offset in larger aggregates as the geometry of the group forces sperm to swim against one another. The result is a non-monotonic relationship between aggregate size and average velocity with both a theoretically predicted and empirically observed optimum of six to seven sperm per aggregate. To understand the role of sexual selection in driving these sperm group dynamics, we compared two sister-species with divergent mating systems. We find that sperm of Peromyscus maniculatus (highly promiscuous), which have evolved under intense competition, form optimal-sized aggregates more often than sperm of Peromyscus polionotus (strictly monogamous), which lack competition. Our combined mathematical and experimental study of coordinated sperm movement reveals the importance of geometry, motion and group size on sperm velocity and suggests how these physical variables interact with evolutionary selective pressures to regulate cooperation in competitive environments. PMID:25056618

  9. Sodium affects the sperm motility in the European eel.

    PubMed

    Vílchez, M Carmen; Morini, Marina; Peñaranda, David S; Gallego, Víctor; Asturiano, Juan F; Pérez, Luz

    2016-08-01

    The role of seminal plasma sodium and activation media sodium on sperm motility was examined by selectively removing the element from these two media, in European eel sperm. Sperm size (sperm head area) was also measured using an ASMA (Automated Sperm Morphometry Analyses) system, in the different conditions. Intracellular sodium [Na(+)]i was quantitatively analyzed by first time in the spermatozoa from a marine fish species. Measurement of [Na(+)]i was done before and after motility activation, by Flow Cytometry, using CoroNa Green AM as a dye. Sperm motility activation induced an increase in [Na(+)]i, from 96.72mM in quiescent stage to 152.21mM post-activation in seawater. A significant decrease in sperm head area was observed post-activation in seawater. There was a notable reduction in sperm motility when sodium was removed from the seminal plasma, but not when it was removed from the activation media. Sodium removal was also linked to a significant reduction in sperm head area in comparison to the controls. Our results indicate that the presence of the ion Na(+) in the seminal plasma (or in the extender medium) is necessary for the preservation of sperm motility in European eel, probably because it plays a role in maintaining an appropriate sperm cell volume in the quiescent stage of the spermatozoa.

  10. Methods of sperm DNA extraction for genetic and epigenetic studies.

    PubMed

    Griffin, Jeanine

    2013-01-01

    High quality DNA extractions developed for mammalian somatic cells are ineffective for sperm, due mainly to the high degree of nuclear compaction in sperm. The highly specialized nuclear proteins in sperm create a chromatin structure that is at least six times denser than histone bound DNA. Unlike somatic cells, sperm DNA is highly compacted by the replacement of histones with sperm-specific low molecular weight proteins called protamines. Both the protamines and the disulfide bridges formed within and between protamines inhibit the extraction of sperm DNA by standard techniques used for somatic cells. Here we describe the guanidine thiocyanate method reported by Hossain with additional modifications resulting in high molecular weight DNA of high quality with an A260/280 ratio ranging between 1.8 and 2.0 and an A260/230 ratio of 2.0 and greater. The DNA is efficiently digested with restriction enzymes and amplified by PCR.

  11. Sperm as microswimmers - navigation and sensing at the physical limit

    NASA Astrophysics Data System (ADS)

    Kaupp, Ulrich B.; Alvarez, Luis

    2016-11-01

    Many cells and microorganisms have evolved a motility apparatus to explore their surroundings. For guidance, these biological microswimmers rely on physical and chemical cues that are transduced by cellular pathways into directed movement - a process called taxis. Only few biological microswimmers have been studied as detailed as sperm from sea urchins. Sperm and eggs are released into the seawater. To enhance the chances of fertilization, eggs release chemical factors - called chemoattractants - that establish a chemical gradient and, thereby, guide sperm to the egg. Sea urchin sperm constitute a unique model system for understanding cell navigation at every level: from molecules to cell behaviours. We will outline the chemotactic signalling pathway of sperm from the sea urchin Arbacia punctulata and discuss how signalling controls navigation in a chemical gradient. Finally, we discuss recent insights into sperm chemotaxis in three dimensions (3D).

  12. Influence of staining and sampling procedures on goat sperm morphometry using the Sperm Class Analyzer.

    PubMed

    Hidalgo, Manuel; Rodríguez, Inmaculada; Dorado, Jesús

    2006-09-01

    Computer-assisted sperm morphometry analysis (ASMA) has improved the assessment of sperm morphology, but the results depend on the use of adequate sampling and staining procedures of spermatozoa from individual species. In this study, the Sperm Class Analyzer ASMA system was used for the morphometric analysis of goat sperm heads. Semen samples, obtained from four bucks, were used to evaluate the influence of three staining procedures (Diff-Quik, Hemacolor and Harris' Haematoxylin) on the accuracy of image processing and sperm morphometry, the effect of the number of cells analysed and the repeatability of the method. These experiments were performed to obtain objective, accurate and reliable sperm morphometric measurements of goat spermatozoa. Diff-Quik and Harris' Haematoxylin were significantly (p<0.05) more accurate than Hemacolor. However, Diff-Quik obtained the highest proportion of correctly analysed sperm heads (86.06%) and the lowest coefficients of variation on the image processing and morphometric measurements. The staining methods affected significantly the sperm dimensions (p<0.001) with increased values from Diff-Quik than Hemacolor and Harris' Haematoxylin, respectively (Diff-Quik>Hemacolor>Harris' Haematoxylin). No differences in morphometric parameters were found when 100, 150, 175 or 200 spermatozoa were analysed. The repeatability of results obtained was very high since no differences were found when measuring the same sperm on multiple attempts. In conclusion, to obtain objective, accurate and repeatable sperm morphometric measurements by the Sperm Class Analyzer system in goats, the analysis of 100 spermatozoa from slides which have been previously stained with Diff-Quik is recommended.

  13. Acrosomo-nuclear syndrome in canine sperm.

    PubMed

    Hrudka, F

    1983-01-01

    An acrosomo-nuclear syndrome in sperm of an infertile semicryptorchid dog is described. Based on an EM study of developing and mature sperm the syndrome is defined by simultaneous occurrence of these symptoms: 1) intranuclear inclusions of acrosomal origin, 2) maldifferentiated apical segment of acrosome, 3) intraacrosomal inclusions of Sertoli cell origin, 4) characteristic change of nuclear shape and 5) retained cytoplasmic droplet. The cause of the syndrome and possibility of a transfer of somatic factors are discussed.

  14. Assessment of sperm DNA fragmentation in stallion (Equus caballus) and donkey (Equus asinus) using the sperm chromatin dispersion test.

    PubMed

    Cortés-Gutiérrez, E I; Crespo, F; Serres-Dalmau, C; Gutiérrez de las Rozas, A L; Dávila-Rodríguez, M I; López-Fernández, C; Gósalvez, J

    2009-10-01

    Sperm DNA fragmentation (sDF) is an important parameter to assessing sperm quality. Information about sperm quality is not available for donkeys, especially in some breeds at risk of extinction. The objectives of this research were to test the four commercial variants of sperm chromatin dispersion test (SCD; sperm Halomax test), originally developed to assess sDF in boars, bulls, rams and stallions, in order to scrutinize their applicability in the study of sDF in a donkey breed at risk of extinction (Zamorano-Leonesa), for which there is no specific test available to analyze sperm at present. Only the SCD test, originally developed for stallions, produced stable and consistent results, and was deemed suitable to assess DNA fragmentation in sperm samples from donkeys. Image analysis was used to compare differences between the SCD methodology applied to stallion and donkey semen samples processed under the same experimental conditions. The extent of SCD in the SCD test was approximately 20% lower in donkey sperm than in stallion sperm. Yet, the ratio of chromatin sperm dispersion achieved in fragmented and unfragmented nuclei did not differ significantly between species. These data suggest that a similar protein depletion treatment can cause differences in protein removal in equivalent cells from different species and that sperm chromatin may be organized differently in stallions and donkeys.

  15. Controlling subcellular delivery to optimize therapeutic effect

    PubMed Central

    Mossalam, Mohanad; Dixon, Andrew S; Lim, Carol S

    2010-01-01

    This article focuses on drug targeting to specific cellular organelles for therapeutic purposes. Drugs can be delivered to all major organelles of the cell (cytosol, endosome/lysosome, nucleus, nucleolus, mitochondria, endoplasmic reticulum, Golgi apparatus, peroxisomes and proteasomes) where they exert specific effects in those particular subcellular compartments. Delivery can be achieved by chemical (e.g., polymeric) or biological (e.g., signal sequences) means. Unidirectional targeting to individual organelles has proven to be immensely successful for drug therapy. Newer technologies that accommodate multiple signals (e.g., protein switch and virus-like delivery systems) mimic nature and allow for a more sophisticated approach to drug delivery. Harnessing different methods of targeting multiple organelles in a cell will lead to better drug delivery and improvements in disease therapy. PMID:21113240

  16. Dynamics of sperm transfer in the ant Leptothorax gredleri

    NASA Astrophysics Data System (ADS)

    Oppelt, Angelika; Heinze, Jürgen

    2007-09-01

    Mating tactics differ remarkably between and within species of social Hymenoptera (bees, wasps, ants) concerning, e.g., mating frequencies, sperm competition, and the degree of male sperm limitation. Although social Hymenoptera might, therefore, potentially be ideal model systems for testing sexual selection theory, the dynamics of mating and sperm transfer have rarely been studied in species other than social bees, and basic information needed to draw conclusions about possible sperm competition and female choice is lacking. We investigated sperm transfer in the ant Leptothorax gredleri, a species in which female sexuals attract males by “female calling.” The analysis of 38 female sexuals fixed immediately or up to 7 days after copulation with a single male each revealed that the sperm is transferred into the female bursa copulatrix embedded in a gelatinous mass, presumably a spermatophore. Sperm cells rapidly start to migrate from the tip of the spermatophore towards the spermatheca, but transfer is drastically slowed down by an extreme constriction of the spermathecal duct, through which sperm cells have to pass virtually one by one. This results in the spermatheca being filled only between one and several hours after mating. During this time, the posterior part of the spermatophore seals the junction between bursa copulatrix and spermathecal duct and prevents sperm loss. The prolonged duration of sperm transfer might allow female sexuals to chose between ejaculates and explain previously reported patterns of single paternity of the offspring of multiply mated queens.

  17. The application of in vitro sperm competition test to evaluate the impact of ZP-derived peptides on fertilization capacity of cat sperm.

    PubMed

    Niu, Yuyu; Greube, Alexa; Ji, Weizhi; Jewgenow, Katarina

    2006-09-01

    The present study aimed to establish a sensitive in vitro assay to assess the binding capacity of cat spermatozoa. Cat oocytes and epididymal sperm cells were isolated from gonads and cultured for in vitro fertilization. Before fertilization, the sperm cells were incubated either in 10 microM green dye Fluo-3-AM or 10 microM orange dye CellTracker Orange CMTMR (Molecular Probes), respectively. After removing the dyes by washing, sperm cells stained with each dye were added to medium drops containing oocytes in various proportions and cultured for 16 h at 37 degrees C, 5% CO(2). The oocytes were examined using fluorescence microscopy. Sperm bound to oocytes, and stained with different colors, were counted. When fresh epididymal sperm were mixed in at a specific proportion, the number of sperm bound to the zona pellucida (ZP) of oocytes reflected the proportion of differently colored sperm in the medium. This indicated that neither dye influenced the binding capacity of cat sperm. Mixing fresh and cryopreserved sperm, however, resulted in a higher number of fresh sperm bound to the oocyte surface in comparison to frozen-thawed sperm. Also, the pre-incubation of cat sperm cells with ZP derived peptide reduced the sperm binding capacity by 40%. In conclusion, the presented sperm competition assay allows assessment of fertilizing capacity of cat spermatozoa in vitro when a mixture of two different populations is used. The applied supravital fluorescence dyes do not affect motility and binding capacity of sperm cells and were clearly distinguishable under fluorescence microscopy. We demonstrate that the assay can be used to study the impact of sperm treatment, such as cryopreservation or pre-incubation in bioactive peptides, on fertilizing capacity.

  18. Copper: a biphasic regulator of caprine sperm forward progression.

    PubMed

    Roy, Debarun; Dey, Souvik; Majumder, Gopal Chandra; Bhattacharyya, Debdas

    2014-02-01

    Copper is essential for spermatogenesis and its presence has been demonstrated in male and female reproductive fluids in several mammalian species. However, little is known about the physiological significance of this trace element in the regulation of forward progression of mammalian sperm cells which is essential for sperm fertility potential in vivo. The purpose of this investigation was to determine the physiological role of the bivalent copper ion (Cu(2+)) on mammalian sperm forward motility using a chemically-defined medium and caprine cauda epididymal sperm model. Sperm forward motility was significantly enhanced by Cu(2+) in a dose-dependent manner; maximal activation (approx 20%) was noted at the 5 µM level of the metal. Above 10 µM Cu(2+) sperm motility decreased, showing that Cu(2+) exerts a biphasic regulation on sperm motility. These findings have been confirmed using a spectrophotometric motility assay, an objective method of motility analysis. At lower concentrations (up to 5 µM), copper enhanced sperm membrane lipid peroxidation as well as the level of intra-sperm cyclic adenosine mono phosphate (c-AMP), but at a higher level it caused marked inhibition of both of the biochemical parameters. The observed correlation of Cu(2+)-dependent biphasic modulation of sperm membrane lipid peroxidation and intrasperm c-AMP with sperm forward motility is consistent with the view that Cu(2+) regulation of sperm motility is mediated by membrane lipid peroxidation, which in turn modulates the level of intra-sperm c-AMP, a well-known activator of sperm motility.

  19. Fluid flow and sperm guidance: a simulation study of hydrodynamic sperm rheotaxis

    PubMed Central

    Ishimoto, Kenta; Gaffney, Eamonn A.

    2015-01-01

    How does a sperm find its way? The study of guidance cues has fascinated sperm biologists and in particular the prospect of rheotaxis, that is a fluid flow orienting the direction of sperm swimming, has been the subject of extensive recent study, as readily motivated by the prospect that such guidance may be active in the mammalian female reproductive tract. For instance, it has been hypothesized that helical sperm flagellar beating is necessary for such guidance, whereas there is an extensive diversity of flagellar beating patterns, with planar sperm beating readily observed in human cells for example. In particular, such cells will not be guided by fluid flow according to hypothesized mechanisms for rheotaxis presented thus far. Here, using simulation methods, we investigate rheotaxis for a wide range of flagellar beat patterns. Providing the virtual sperm firstly does not possess a tightly circling trajectory in the absence of a background flow and secondly, remains within a region of low shear to prevent being washed away by the background flow, rheotaxis is generally observed with the sperm swimming into the flow together with a possible transverse velocity. Tight circling sperm motility, as observed in select hyperactivated sperm and CatSper mutants, is predicted to disrupt the rheotactic response, whereas confinement to low shear regions generally requires boundary accumulation, thus introducing subtleties in the relationship between rheotactic behaviours and the flagellar waveform and sperm characteristics. Nonetheless, such predictions suggest such rheotactic guidance may be more common and robust than previously thought, and we document simple criteria for the presence of rheotaxis that are consistent with our simulations and understanding, as well as reported observations to date. PMID:25878133

  20. Genetic sperm defects.

    PubMed

    Chenoweth, Peter J

    2005-08-01

    Genetic sperm defects are specific sperm defects, which have been shown to have a genetic mode of transmission. Such genetic linkage, either direct or indirect, has been associated with a number of sperm defects in different species, with this number increasing with improved diagnostic capabilities. A number of sperm defects, which have proven or suspected genetic modes of transmission are discussed herein, with particular emphasis on cattle. These include: 1. Acrosome defects (knobbed, ruffled and incomplete); 2. Head defects (abnormal condensation, decapitated, round head, rolled head, nuclear crest); 3. Midpiece abnormalities ("Dag" defect, "corkscrew" defect, "pseudo-droplet" defect); 4. Tail defects ("tail stump" defect, primary ciliary dyskinesia).

  1. Novel Flow Cytometry Analyses of Boar Sperm Viability: Can the Addition of Whole Sperm-Rich Fraction Seminal Plasma to Frozen-Thawed Boar Sperm Affect It?

    PubMed Central

    Díaz, Rommy; Boguen, Rodrigo; Martins, Simone Maria Massami Kitamura; Ravagnani, Gisele Mouro; Leal, Diego Feitosa; Oliveira, Melissa de Lima; Muro, Bruno Bracco Donatelli; Parra, Beatriz Martins; Meirelles, Flávio Vieira; Papa, Frederico Ozanan; Dell’Aqua, José Antônio; Alvarenga, Marco Antônio; Moretti, Aníbal de Sant’Anna; Sepúlveda, Néstor

    2016-01-01

    Boar semen cryopreservation remains a challenge due to the extension of cold shock damage. Thus, many alternatives have emerged to improve the quality of frozen-thawed boar sperm. Although the use of seminal plasma arising from boar sperm-rich fraction (SP-SRF) has shown good efficacy; however, the majority of actual sperm evaluation techniques include a single or dual sperm parameter analysis, which overrates the real sperm viability. Within this context, this work was performed to introduce a sperm flow cytometry fourfold stain technique for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential. We then used the sperm flow cytometry fourfold stain technique to study the effect of SP-SRF on frozen-thawed boar sperm and further evaluated the effect of this treatment on sperm movement, tyrosine phosphorylation and fertility rate (FR). The sperm fourfold stain technique is accurate (R2 = 0.9356, p > 0.01) for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential (IPIAH cells). Centrifugation pre-cryopreservation was not deleterious (p > 0.05) for any analyzed variables. Addition of SP-SRF after cryopreservation was able to improve total and progressive motility (p < 0.05) when boar semen was cryopreserved without SP-SRF; however, it was not able to decrease tyrosine phosphorylation (p > 0.05) or improve IPIAH cells (p > 0.05). FR was not (p > 0.05) statistically increased by the addition of seminal plasma, though females inseminated with frozen-thawed boar semen plus SP-SRF did perform better than those inseminated with sperm lacking seminal plasma. Thus, we conclude that sperm fourfold stain can be used to simultaneously evaluate plasma and acrosomal membrane integrity and mitochondrial membrane potential, and the addition of SP-SRF at thawed boar semen cryopreserved in absence of SP-SRF improve its total and progressive motility. PMID:27529819

  2. Imaging trace element distributions in single organelles and subcellular features

    SciTech Connect

    Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; Rose, Volker; Vogt, Stefan; El-Muayed, Malek

    2016-02-25

    The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro- and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cd (which some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators.We find it could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies.

  3. Imaging trace element distributions in single organelles and subcellular features

    PubMed Central

    Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; Rose, Volker; Vogt, Stefan; El-Muayed, Malek

    2016-01-01

    The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro- and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cd (which some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators. It could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies. PMID:26911251

  4. Imaging trace element distributions in single organelles and subcellular features

    NASA Astrophysics Data System (ADS)

    Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; Rose, Volker; Vogt, Stefan; El-Muayed, Malek

    2016-02-01

    The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro- and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cd (which some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators. It could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies.

  5. Intracellular pH in Sperm Physiology

    PubMed Central

    Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L.; Darszon, Alberto

    2014-01-01

    Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca2+ channel; Slo3, a K+ channel; the sperm-specific Na+/H+ exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction. PMID:24887564

  6. Mammalian Sperm Motility: Observation and Theory

    NASA Astrophysics Data System (ADS)

    Gaffney, E. A.; Gadêlha, H.; Smith, D. J.; Blake, J. R.; Kirkman-Brown, J. C.

    2011-01-01

    Mammalian spermatozoa motility is a subject of growing importance because of rising human infertility and the possibility of improving animal breeding. We highlight opportunities for fluid and continuum dynamics to provide novel insights concerning the mechanics of these specialized cells, especially during their remarkable journey to the egg. The biological structure of the motile sperm appendage, the flagellum, is described and placed in the context of the mechanics underlying the migration of mammalian sperm through the numerous environments of the female reproductive tract. This process demands certain specific changes to flagellar movement and motility for which further mechanical insight would be valuable, although this requires improved modeling capabilities, particularly to increase our understanding of sperm progression in vivo. We summarize current theoretical studies, highlighting the synergistic combination of imaging and theory in exploring sperm motility, and discuss the challenges for future observational and theoretical studies in understanding the underlying mechanics.

  7. Molecular Cloning of Spergen-4, Encoding a Spermatogenic Cell-Specific Protein Associated with Sperm Flagella and the Acrosome Region in Rat Spermatozoa.

    PubMed

    Howida, Ali; Salaheldeen, Elsaid; Iida, Hiroshi

    2016-04-01

    We used a differential display in combination with complementary DNA (cDNA) cloning approach to isolate a novel rat gene LOC690919 with an open reading frame of 1227-length nucleotides encoding a protein of 409 amino acids. This gene was designated as Spergen-4 (a spermatogenic cell-specific gene-4). Spergen-4 mRNA was highly expressed in testis, and its expression was detected in rat testis starting at three weeks of postnatal development and persisting up to adulthood. Mouse and human orthologs, which lack N-terminal 77 amino acid residues of rat Spegen-4, were found in the database. Immunofluorescence microscopy and immunoblot analysis demonstrated that Spergen-4 was not expressed in spermatogonia, spermatocytes, and round spermatids, but was restrictedly detected at sperm head, cytoplasm, and developing flagella of elongated spermatids in rat testis. In mature spermatozoa, Spergen-4 was detected at the acrosome region as well as the principal piece of flagella. Spergen-4 immunosignal disappeared from sperm heads on acrosome reaction induced by progesterone. These data suggest that Spergen-4 integrated into elongated spermatids during spermiogenesis serves as a constituent for acrosome region and flagella of rat spermatozoa.

  8. Co-administration of ginseng and ciprofloxacin ameliorates epididymo-orchitis induced alterations in sperm quality and spermatogenic cells apoptosis following infection in rats.

    PubMed

    Eskandari, M; Ghalyanchi Langeroudi, A; Zeighami, H; Rostami, A; Kazemi, M; Eyni, H; Shokri, S

    2017-04-01

    Korean red ginseng (KRG) may be a beneficial adjuvant along with ciprofloxacin to ameliorate devastating effects of epididymo-orchitis (EO) on male fertility. This study intends to assay the effects of KRG and ciprofloxacin on sperm quality and spermatogenic cells apoptosis in EO rats. We divided 54 adult rats into nine groups (n = 6 rats per group): control (CO), sham-operated (SH), EO (E); ciprofloxacin (C), EO-ciprofloxacin (EC), KRG (G), EO-KRG (EG), ciprofloxacin-KRG (CG) and EO-ciprofloxacin-KRG (ECG). We administered ciprofloxacin and KRG 48 hr after the Escherichia coli (E. coli) injection for 10 days. Bilateral orchiectomy was performed after one sperm cycle (14 days) following the last treatment with ciprofloxacin and KRG. Total and progressive motility of E, C and EC groups decreased. However, motility is improved in CG and ECG in comparison with these groups. The E group induced negative changes in the architecture of testes tissue and dramatic increase in apoptosis indices. Interestingly, co-administration of ciprofloxacin and KRG has dramatically improved Miller's and Johnsen's scores and decreased the apoptosis indices of animals in the ECG group. Combined treatment of ciprofloxacin and KRG may improve the quality of spermatozoa and attenuated apoptosis indices in the ECG group.

  9. Calcium signaling and the MAPK cascade are required for sperm activation in Caenorhabditis elegans.

    PubMed

    Liu, Zhiyu; Wang, Bin; He, Ruijun; Zhao, Yanmei; Miao, Long

    2014-02-01

    In nematode, sperm activation (or spermiogenesis), a process in which the symmetric and non-motile spermatids transform into polarized and crawling spermatozoa, is critical for sperm cells to acquire fertilizing competence. SPE-8 dependent and SPE-8 independent pathways function redundantly during sperm activation in both males and hermaphrodites of Caenorhabditis elegans. However, the downstream signaling for both pathways remains unclear. Here we show that calcium signaling and the MAPK cascade are required for both SPE-8 dependent and SPE-8 independent sperm activation, implying that both pathways share common downstream signaling components during sperm activation. We demonstrate that activation of the MAPK cascade is sufficient to activate spermatids derived from either wild-type or spe-8 group mutant males and that activation of the MAPK cascade bypasses the requirement of calcium signal to induce sperm activation, indicating that the MAPK cascade functions downstream of or parallel with the calcium signaling during sperm activation. Interestingly, the persistent activation of MAPK in activated spermatozoa inhibits Major Sperm Protein (MSP)-based cytoskeleton dynamics. We demonstrate that MAPK plays dual roles in promoting pseudopod extension during sperm activation but also blocking the MSP-based, amoeboid motility of the spermatozoa. Thus, though nematode sperm are crawling cells, morphologically distinct from flagellated sperm, and the molecular machinery for motility of amoeboid and flagellated sperm is different, both types of sperm might utilize conserved signaling pathways to modulate sperm maturation.

  10. Imaging Subcellular Structures in the Living Zebrafish Embryo.

    PubMed

    Engerer, Peter; Plucinska, Gabriela; Thong, Rachel; Trovò, Laura; Paquet, Dominik; Godinho, Leanne

    2016-04-02

    In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

  11. Flow cytometry application in the assessment of sperm DNA integrity of men with asthenozoospermia.

    PubMed

    Piasecka, M; Gaczarzewicz, D; Laszczyńska, M; Starczewski, A; Brodowska, A

    2007-01-01

    Sperm genomic integrity and ultrastructural features of ejaculated spermatozoa contributing to the assessment of gamete fertility potential in patients with asthenozoospermia are discussed. The proportion of TUNEL-positive cells was significantly higher in the semen of patients with low sperm motility (n=40; p<0.01) as compared to men with normal sperm motility (n=54). Sperm DNA fragmentation negatively correlated (n=94) with sperm motility, sperm concentration, and integrity of the sperm cellular membrane (HOS-test). Two categories of patients were distinguished: (1) patients (23 out of 94 subjects) with < or = 4% of TUNEL-positive cells and (2) patients (71 subjects) with 4% of TUNEL-positive cells. A significant difference was noted in the sperm motility and HOS-test results between patients from both groups. Large numbers of immature spermatozoa with extensive cytoplasmic retention, ultrastructural chromatin and midpiece abnormalities, and conglomerates containing sperm fragments were present more frequently in the semen of asthenozoospermic subjects with >4% of TUNEL-positive sperm cells. Low sperm motility seems to be accompanied by serious defects of gamete chromatin expressed as diminished sperm genomic integrity and abnormal DNA condensation and by defects of sperm midpiece. These abnormalities may reflect developmental failure during the spermatogenic remodeling process. The DNA fragmentation test may be considered as an additional assay for the evaluation of spermatozoa beside standard analysis and taken together with electron microscopy may help to determine the actual number of "healthy" spermatozoa thereby playing an important role during diagnosis and treatment of male infertility.

  12. An interdisciplinary systems approach to study sperm physiology and evolution.

    PubMed

    Shi, Linda Z; Nascimento, Jaclyn; Botvinick, Elliot; Durrant, Barbara; Berns, Michael W

    2011-01-01

    Optical trapping is a noninvasive biophotonic tool that has been developed to study the physiological and biomechanical properties of cells. The custom-designed optical system is built to direct near-infrared laser light into an inverted microscope to create a single-point three-dimensional gradient laser trap at the microscope focal point. A real-time automated tracking and trapping system (RATTS) is described that provides a remote user-friendly robotic interface. The combination of laser tweezers, fluorescent imaging, and RATTS can measure sperm swimming speed and swimming force simultaneously with mitochondrial membrane potential (MMP). The roles of two sources of adenosine triphosphate in sperm motility/energetics are studied: oxidative phosphorylation, which occurs in the mitochondria located in the sperm midpiece, and glycolysis, which occurs along the length of the sperm tail (flagellum). The effects of glucose, oxidative phosphorylation inhibitors, and glycolytic inhibitors on human sperm motility are studied. This combination of photonic physical and engineering tools has been used to examine the evolutionary effect of sperm competition in primates. The results demonstrate a correlation between mating type and sperm motility: sperm from polygamous (multi-partner) primate species swim faster and with greater force than sperm from polygynous (single partner) primate species. In summary, engineering and biological systems are combined to provide a powerful interdisciplinary approach to study the complex biological systems that drive the sperm toward the egg.

  13. Sexing mammalian sperm--intertwining of commerce, technology, and biology.

    PubMed

    Seidel, George E

    2003-12-15

    Sperm from many mammalian species can be sexed by flow cytometry/cell sorting at about 90% accuracy without damaging them unduly. However, because sperm are evaluated one at a time, in series, the number of sexed sperm produced per unit time is limited. Furthermore, the equipment required currently is expensive, in the order of 300,000 US dollars per machine. Despite these limitations, commercialization of this technology has begun with bovine semen, in part by inseminating cattle with relatively low number of sperms. No other approach to sexing sperm in any practical way is likely to be available within the next few years. The constraints for commercial application of sexed sperm in cattle can be somewhat lowered fertility, the high costs of equipment and skilled personnel, and costs of intellectual property such as licensing fees and royalty payments. Most economic analyses indicate that farmers can afford to pay 10-20 US dollars more per dose of sexed sperm than unsexed sperm if costs must be recouped by selling milk or meat. When the product is breeding stock or for certain niche applications, a higher price for sexed semen may be justified. Sexed sperm will be used more broadly in cattle only when improved production and/or efficiency can compensate for the extra costs of purchasing sexed sperm.

  14. Ion channels: Key elements in sea urchin sperm physiology

    NASA Astrophysics Data System (ADS)

    Darszon, Alberto; de De Latorre, Lucia; Vargas, Irma; Liévano, Arturo; Beltrán, Carmen; Santi, Celia; Labarca, Pedro; Zapata, Otilia

    1995-08-01

    Ion channels are deeply involved in sea urchin sperm activation, motility, chemotaxis and in the acrosome reaction. Unraveling ion channel function and regulation in sperm behavior has required a combination of complementary approaches since spermatozoa are very tiny cells. Planar bilayer and patch clamp techniques have allowed us to detect, for the first time, the activity of single channels in the plasma membrane of these cells. Unlike intact sperm, swollen sperm can be much more easily patch clamped and single channel activity recorded. These techniques, together with studies of membrane potential, intracellular Ca2+ and pH in whole sperm, have established the presence of K+, Ca2+, and Cl- channels in this cell. The strategies developed to study sea urchin sperm channels are applicable to mammalian spermatozoa. We recently detected a Ca2+ channel resembling one found in S. purpuratus sperm in planar bilayers containing mouse sperm plasma membranes. The presence of this Ca2+ channel in such diverse species suggests it is important in sperm function.

  15. Biomechanics of subcellular structures by non-invasive Brillouin microscopy

    PubMed Central

    Antonacci, Giuseppe; Braakman, Sietse

    2016-01-01

    Cellular biomechanics play a pivotal role in the pathophysiology of several diseases. Unfortunately, current methods to measure biomechanical properties are invasive and mostly limited to the surface of a cell. As a result, the mechanical behaviour of subcellular structures and organelles remains poorly characterised. Here, we show three-dimensional biomechanical images of single cells obtained with non-invasive, non-destructive Brillouin microscopy with an unprecedented spatial resolution. Our results quantify the longitudinal elastic modulus of subcellular structures. In particular, we found the nucleoli to be stiffer than both the nuclear envelope (p < 0.0001) and the surrounding cytoplasm (p < 0.0001). Moreover, we demonstrate the mechanical response of cells to Latrunculin-A, a drug that reduces cell stiffness by preventing cytoskeletal assembly. Our technique can therefore generate valuable insights into cellular biomechanics and its role in pathophysiology. PMID:27845411

  16. Biomechanics of subcellular structures by non-invasive Brillouin microscopy

    NASA Astrophysics Data System (ADS)

    Antonacci, Giuseppe; Braakman, Sietse

    2016-11-01

    Cellular biomechanics play a pivotal role in the pathophysiology of several diseases. Unfortunately, current methods to measure biomechanical properties are invasive and mostly limited to the surface of a cell. As a result, the mechanical behaviour of subcellular structures and organelles remains poorly characterised. Here, we show three-dimensional biomechanical images of single cells obtained with non-invasive, non-destructive Brillouin microscopy with an unprecedented spatial resolution. Our results quantify the longitudinal elastic modulus of subcellular structures. In particular, we found the nucleoli to be stiffer than both the nuclear envelope (p < 0.0001) and the surrounding cytoplasm (p < 0.0001). Moreover, we demonstrate the mechanical response of cells to Latrunculin-A, a drug that reduces cell stiffness by preventing cytoskeletal assembly. Our technique can therefore generate valuable insights into cellular biomechanics and its role in pathophysiology.

  17. Arabidopsis myosin XI sub-domains homologous to the yeast myo2p organelle inheritance sub-domain target subcellular structures in plant cells

    PubMed Central

    Sattarzadeh, Amirali; Schmelzer, Elmon; Hanson, Maureen R.

    2013-01-01

    Myosin XI motor proteins transport plant organelles on the actin cytoskeleton. The Arabidopsis gene family that encodes myosin XI has 13 members, 12 of which have sub-domains within the tail region that are homologous to well-characterized cargo-binding domains in the yeast myosin V myo2p. Little is presently known about the cargo-binding domains of plant myosin XIs. Prior experiments in which most or all of the tail regions of myosin XIs have been fused to yellow fluorescent protein (YFP) and transiently expressed have often not resulted in fluorescent labeling of plant organelles. We identified 42 amino-acid regions within 12 Arabidopsis myosin XIs that are homologous to the yeast myo2p tail region known to be essential for vacuole and mitochondrial inheritance. A YFP fusion of the yeast region expressed in plants did not label tonoplasts or mitochondria. We investigated whether the homologous Arabidopsis regions, termed by us the “PAL” sub-domain, could associate with subcellular structures following transient expression of fusions with YFP in Nicotiana benthamiana. Seven YFP::PAL sub-domain fusions decorated Golgi and six were localized to mitochondria. In general, the myosin XI PAL sub-domains labeled organelles whose motility had previously been observed to be affected by mutagenesis or dominant negative assays with the respective myosins. Simultaneous transient expression of the PAL sub-domains of myosin XI-H, XI-I, and XI-K resulted in inhibition of movement of mitochondria and Golgi. PMID:24187546

  18. Evaluation of Lasting Effects of Heat Stress on Sperm Profile and Oxidative Status of Ram Semen and Epididymal Sperm

    PubMed Central

    Hamilton, Thais Rose dos Santos; Mendes, Camilla Mota; de Castro, Letícia Signori; de Assis, Patrícia Monken; Siqueira, Adriano Felipe Perez; Delgado, Juliana de Carvalho; Goissis, Marcelo Demarchi; Muiño-Blanco, Teresa; Cebrián-Pérez, José Álvaro; Nichi, Marcílio; Visintin, José Antonio; Assumpção, Mayra Elena Ortiz D'Ávila

    2016-01-01

    Higher temperatures lead to an increase of testicular metabolism that results in spermatic damage. Oxidative stress is the main factor responsible for testicular damage caused by heat stress. The aim of this study was to evaluate lasting effects of heat stress on ejaculated sperm and immediate or long-term effects of heat stress on epididymal sperm. We observed decrease in motility and mass motility of ejaculated sperm, as well as an increase in the percentages of sperm showing major and minor defects, damaged plasma and acrosome membranes, and a decrease in the percentage of sperm with high mitochondrial membrane potential in the treated group until one spermatic cycle. An increased enzymatic activity of glutathione peroxidase and an increase of stressed cells were observed in ejaculated sperm of the treated group. A decrease in the percentage of epididymal sperm with high mitochondrial membrane potential was observed in the treated group. However, when comparing immediate and long-term effects, we observed an increase in the percentage of sperm with low mitochondrial membrane potential. In conclusion, testicular heat stress induced oxidative stress that led to rescuable alterations after one spermatic cycle in ejaculated sperm and also after 30 days in epididymal sperm. PMID:26881013

  19. Sperm navigation in complex environments

    NASA Astrophysics Data System (ADS)

    Olson, Sarah

    2016-11-01

    Sperm can swim in a variety of environments, interacting with chemicals and other proteins in the fluid. Some of these extra proteins or cells may act as friction, possibly preventing or enhancing forward progression of swimmers. The homogenized fluid flow is assumed to be governed by the incompressible Brinkman equation, where a friction term with a resistance parameter represents a sparse array of obstacles. Representing the swimmers with a centerline approximation, we employ regularized fundamental solutions to investigate swimming speeds, trajectories, and interactions of swimmers. Asymmetric waveforms due to an increase in flagellar calcium is known to be important for sperm to reach and fertilize the egg. The trajectories of hyperactivated swimmers are found to have a decreased path curvature. Although attraction of two swimmers is more efficient in the Stokes regime, we find that attraction does not occur for larger resistance. Additionally, we study interactions of swimmers in a channel. NSF DMS 1413110.

  20. Magnetic propulsion of robotic sperms at low-Reynolds number

    NASA Astrophysics Data System (ADS)

    Khalil, Islam S. M.; Fatih Tabak, Ahmet; Klingner, Anke; Sitti, Metin

    2016-07-01

    We investigate the microswimming behaviour of robotic sperms in viscous fluids. These robotic sperms are fabricated from polystyrene dissolved in dimethyl formamide and iron-oxide nanoparticles. This composition allows the nanoparticles to be concentrated within the bead of the robotic sperm and provide magnetic dipole, whereas the flexibility of the ultra-thin tail enables flagellated locomotion using magnetic fields in millitesla range. We show that these robotic sperms have similar morphology and swimming behaviour to those of sperm cells. Moreover, we show experimentally that our robotic sperms swim controllably at an average speed of approximately one body length per second (around 125 μm s-1), and they are relatively faster than the microswimmers that depend on planar wave propulsion in low-Reynolds number fluids.

  1. Subcellular localization of FOXO3a as a potential biomarker of response to combined treatment with inhibitors of PI3K and autophagy in PIK3CA-mutant cancer cells

    PubMed Central

    Young, Chan Kim; Hwan, Yun Kim; Ju, Woong; Cheol, Seung Kim

    2017-01-01

    Autophagy is the process of lysosome-mediated degradation and recycling that functions as an adaptive survival mechanism during anti-cancer therapy. Aberrant activation of the phosphoinositide-3-kinase (PI3K) pathway frequently occurs in solid tumors, including cervical cancer. However, single-agent PI3K inhibitors show modest anti-tumor efficacy in clinics. To see whether autophagy inhibition improves the efficacy of PI3K inhibitor in PIK3CA-mutant cancer cells, cells were treated with BKM120, a pan-PI3K inhibitor, and the autophagy inhibitor hydroxychloroquine (HCQ). Autophagy inhibition augmented the efficacy of BKM120 depending on PIK3CA-mutant cancer cell type. BKM120 treatment led to the nuclear accumulation of forkhead box O3 (FOXO3a) in Caski and T47D cells, which showed a synergistic effect of BKM120 and HCQ and the strong induction of autophagy. However, most FOXO3a remained in cytoplasm in C33A and ME180 cells, which did not exhibit synergy. These data suggest that BKM120-induced nuclear translocation of FOXO3a might elicit autophagy and be a critical factor determining the synergistic activity of BKM120 and HCQ in PIK3CA-mutant cancer cells. The release of FOXO3a from 14-3-3 by BV02 or 14-3-3 knockdown induced autophagy by BKM120 in C33A cells and sensitized the cells to the combined BKM120 and HCQ treatment, suggesting that cytoplasmic retention of FOXO3a by 14-3-3 even in the presence of BKM120 inhibit autophagy induction and synergistic effect of BKM120 and HCQ combination. Taken together, our study shows that subcellular localization of FOXO3a might be a potential biomarker for predicting response to the combination treatment with PI3K and autophagy inhibitors in PIK3CA-mutant cervical cancer patients. PMID:28036259

  2. Mass-Specific Metabolic Rate Influences Sperm Performance through Energy Production in Mammals.

    PubMed

    Tourmente, Maximiliano; Roldan, Eduardo R S

    2015-01-01

    Mass-specific metabolic rate, the rate at which organisms consume energy per gram of body weight, is negatively associated with body size in metazoans. As a consequence, small species have higher cellular metabolic rates and are able to process resources at a faster rate than large species. Since mass-specific metabolic rate has been shown to constrain evolution of sperm traits, and most of the metabolic activity of sperm cells relates to ATP production for sperm motility, we hypothesized that mass-specific metabolic rate could influence sperm energetic metabolism at the cellular level if sperm cells maintain the metabolic rate of organisms that generate them. We compared data on sperm straight-line velocity, mass-specific metabolic rate, and sperm ATP content from 40 mammalian species and found that the mass-specific metabolic rate positively influences sperm swimming velocity by (a) an indirect effect of sperm as the result of an increased sperm length, and (b) a direct effect independent of sperm length. In addition, our analyses show that species with higher mass-specific metabolic rate have higher ATP content per sperm and higher concentration of ATP per μm of sperm length, which are positively associated with sperm velocity. In conclusion, our results suggest that species with high mass-specific metabolic rate have been able to evolve both long and fast sperm. Moreover, independently of its effect on the production of larger sperm, the mass-specific metabolic rate is able to influence sperm velocity by increasing sperm ATP content in mammals.

  3. Comparison of methods for assessing integrity of equine sperm membranes.

    PubMed

    Foster, M L; Love, C C; Varner, D D; Brinsko, S P; Hinrichs, K; Teague, S; Lacaze, K; Blanchard, T L

    2011-07-15

    Sperm membrane integrity (SMI) is thought to be an important measure of stallion sperm quality. The objective was to compare three methods for evaluating SMI: flow cytometry using SYBR-14/propidium iodide (PI) stain; an automated cell counting device using PI stain; and eosin-nigrosin stain. Raw equine semen was subjected to various treatments containing 20 to 80% seminal plasma in extender, with differing sperm concentrations, to simulate spontaneous loss of SMI. The SMI was assessed immediately, and after 1 and 2 d of cooled storage. Agreement between methods was determined according to Bland-Altman methodology. Eosin-nigrosin staining yielded higher (2%) overall mean values for SMI than did flow cytometry. Flow cytometry yielded higher (6%) overall mean values for SMI than did the automated cell counter. As percentage of membrane-damaged sperm increased, agreement of SMI measurement between methods decreased. When semen contained 50-79% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = -26.9 to 24.3%; i.e., a 51.2% span) than for SMI determined by flow cytometry and the automated cell counter (range = -3.1 to 17.0%; 20.1% span). When sperm populations contained <50% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = -35.9 to 19.0%; 54.9% span) than for SMI determined by flow cytometry and the automated cell counter (range = -11.6 to 28.7%; 40.3% span). We concluded that eosin-nigrosin staining assessments of percent membrane-intact sperm agreed less with flow cytometry when <80% of sperm had intact membranes, whereas automated cell counter assessments of percent membrane-intact sperm agreed less with flow cytometry when <30% of sperm had intact membranes.

  4. Protein kinase C activity in boar sperm.

    PubMed

    Teijeiro, J M; Marini, P E; Bragado, M J; Garcia-Marin, L J

    2017-03-01

    Male germ cells undergo different processes within the female reproductive tract to successfully fertilize the oocyte. These processes are triggered by different extracellular stimuli leading to activation of protein phosphorylation. Protein kinase C (PKC) is a key regulatory enzyme in signal transduction mechanisms involved in many cellular processes. Studies in boar sperm demonstrated a role for PKC in the intracellular signaling involved in motility and cellular volume regulation. Experiments using phorbol 12-myristate 13-acetate (PMA) showed increases in the Serine/Threonine phosphorylation of substrates downstream of PKC in boar sperm. In order to gain knowledge about those cellular processes regulated by PKC, we evaluate the effects of PMA on boar sperm motility, lipid organization of plasma membrane, integrity of acrosome membrane and sperm agglutination. Also, we investigate the crosstalk between PKA and PKC intracellular pathways in spermatozoa from this species. The results presented here reveal a participation of PKC in sperm motility regulation and membrane fluidity changes, which is probably associated to acrosome reaction and to agglutination. Also, we show the existence of a hierarchy in the kinases pathway. Previous works on boar sperm suggest a pathway in which PKA is positioned upstream to PKC and this new results support such model.

  5. Major regulatory mechanisms involved in sperm motility.

    PubMed

    Pereira, Rute; Sá, Rosália; Barros, Alberto; Sousa, Mário

    2017-01-01

    The genetic bases and molecular mechanisms involved in the assembly and function of the flagellum components as well as in the regulation of the flagellar movement are not fully understood, especially in humans. There are several causes for sperm immotility, of which some can be avoided and corrected, whereas other are related to genetic defects and deserve full investigation to give a diagnosis to patients. This review was performed after an extensive literature search on the online databases PubMed, ScienceDirect, and Web of Science. Here, we review the involvement of regulatory pathways responsible for sperm motility, indicating possible causes for sperm immotility. These included the calcium pathway, the cAMP-dependent protein kinase pathway, the importance of kinases and phosphatases, the function of reactive oxygen species, and how the regulation of cell volume and osmolarity are also fundamental components. We then discuss main gene defects associated with specific morphological abnormalities. Finally, we slightly discuss some preventive and treatments approaches to avoid development of conditions that are associated with unspecified sperm immotility. We believe that in the near future, with the development of more powerful techniques, the genetic causes of sperm immotility and the regulatory mechanisms of sperm motility will be better understand, thus enabling to perform a full diagnosis and uncover new therapies.

  6. Major regulatory mechanisms involved in sperm motility

    PubMed Central

    Pereira, Rute; Sá, Rosália; Barros, Alberto; Sousa, Mário

    2017-01-01

    The genetic bases and molecular mechanisms involved in the assembly and function of the flagellum components as well as in the regulation of the flagellar movement are not fully understood, especially in humans. There are several causes for sperm immotility, of which some can be avoided and corrected, whereas other are related to genetic defects and deserve full investigation to give a diagnosis to patients. This review was performed after an extensive literature search on the online databases PubMed, ScienceDirect, and Web of Science. Here, we review the involvement of regulatory pathways responsible for sperm motility, indicating possible causes for sperm immotility. These included the calcium pathway, the cAMP-dependent protein kinase pathway, the importance of kinases and phosphatases, the function of reactive oxygen species, and how the regulation of cell volume and osmolarity are also fundamental components. We then discuss main gene defects associated with specific morphological abnormalities. Finally, we slightly discuss some preventive and treatments approaches to avoid development of conditions that are associated with unspecified sperm immotility. We believe that in the near future, with the development of more powerful techniques, the genetic causes of sperm immotility and the regulatory mechanisms of sperm motility will be better understand, thus enabling to perform a full diagnosis and uncover new therapies. PMID:26680031

  7. Membrane hyperpolarization during human sperm capacitation

    PubMed Central

    López-González, I.; Torres-Rodríguez, P.; Sánchez-Carranza, O.; Solís-López, A.; Santi, C.M.; Darszon, A.; Treviño, C.L.

    2014-01-01

    Sperm capacitation is a complex and indispensable physiological process that spermatozoa must undergo in order to acquire fertilization capability. Spermatozoa from several mammalian species, including mice, exhibit a capacitation-associated plasma membrane hyperpolarization, which is necessary for the acrosome reaction to occur. Despite its importance, this hyperpolarization event has not been adequately examined in human sperm. In this report we used flow cytometry to show that a subpopulation of human sperm indeed undergo a plasma membrane hyperpolarization upon in vitro capacitation. This hyperpolarization correlated with two other well-characterized capacitation parameters, namely an increase in intracellular pH and Ca2+ concentration, measured also by flow cytometry. We found that sperm membrane hyperpolarization was completely abolished in the presence of a high external K+ concentration (60 mM), indicating the participation of K+ channels. In order to identify, which of the potential K+ channels were involved in this hyperpolarization, we used different K+ channel inhibitors including charybdotoxin, slotoxin and iberiotoxin (which target Slo1) and clofilium (a more specific blocker for Slo3). All these K+ channel antagonists inhibited membrane hyperpolarization to a similar extent, suggesting that both members of the Slo family may potentially participate. Two very recent papers recorded K+ currents in human sperm electrophysiologically, with some contradictory results. In the present work, we show through immunoblotting that Slo3 channels are present in the human sperm membrane. In addition, we found that human Slo3 channels expressed in CHO cells were sensitive to clofilium (50 μM). Considered altogether, our data indicate that Slo1 and Slo3 could share the preponderant role in the capacitation-associated hyperpolarization of human sperm in contrast to what has been previously reported for mouse sperm, where Slo3 channels are the main contributors to the

  8. High-throughput flowing upstream sperm sorting in a retarding flow field for human semen analysis.

    PubMed

    Wu, Jen-Kuei; Chen, Peng-Chun; Lin, Yu-Nan; Wang, Chia-Woei; Pan, Li-Chern; Tseng, Fan-Gang

    2017-03-13

    In this paper, we propose a microfluidic device capable of generating a retarding flow field for the sorting and separation of human motile sperm in a high-throughput manner. The proposed sorting/separation process begins with a rapid flow field in a straight-flow zone to carry sperm into a sorting zone to maintain the sperm's mobility. The sorting zone consists of a diffuser-type sperm sorter to differentiate sperm with different motilities based on the flowing upstream nature of human sperm in a retarding flow field. The dead sperm will then be separated from the live ones by passing through a dumbbell flow field to the outlet for disposal. The proposed flowing upstream sperm sorter (FUSS) is designed to imitate the selection mechanism found in the female body when sperm swim into the uterus. The experimental results demonstrate the utility of this device with regard to throughput (approximately 200 000 sperm per minute and a maximum of 200 million cells per mL), efficiency (90% of selected sperm are mobile), and the ability to select sperm with high motility (∼20% of sperm with a velocity exceeding 120 μm s(-1)). The proposed device is suitable for intrauterine insemination as well as in vitro fertilization thanks to the highly efficient sorting process not interfering with the natural function and energy resource of human sperm.

  9. Global subcellular characterization of protein degradation using quantitative proteomics.

    PubMed

    Larance, Mark; Ahmad, Yasmeen; Kirkwood, Kathryn J; Ly, Tony; Lamond, Angus I

    2013-03-01

    Protein degradation provides an important regulatory mechanism used to control cell cycle progression and many other cellular pathways. To comprehensively analyze the spatial control of protein degradation in U2OS osteosarcoma cells, we have combined drug treatment and SILAC-based quantitative mass spectrometry with subcellular and protein fractionation. The resulting data set analyzed more than 74,000 peptides, corresponding to ~5000 proteins, from nuclear, cytosolic, membrane, and cytoskeletal compartments. These data identified rapidly degraded proteasome targets, such as PRR11 and highlighted a feedback mechanism resulting in translation inhibition, induced by blocking the proteasome. We show this is mediated by activation of the unfolded protein response. We observed compartment-specific differences in protein degradation, including proteins that would not have been characterized as rapidly degraded through analysis of whole cell lysates. Bioinformatic analysis of the entire data set is presented in the Encyclopedia of Proteome Dynamics, a web-based resource, with proteins annotated for stability and subcellular distribution.

  10. Changes of sperm quality and hormone receptors in the rat testis after exposure to methamphetamine.

    PubMed

    Nudmamud-Thanoi, Sutisa; Sueudom, Wanvipa; Tangsrisakda, Nareelak; Thanoi, Samur

    2016-10-01

    Methamphetamine (METH) is known to damage neurons and induce psychosis. It can also induce apoptosis in seminiferous tubules and affect sperm quality. The present study was carried out to investigate the effect of a rat model of METH addiction on sperm quality and expression of progesterone receptors (PR) and estrogen receptors (ER) in the testis. Sperm quality parameters including sperm motility, sperm morphology and sperm concentration were examined. Protein and gene expressions PR, ERα and ERβ were studied using immunohistochemistry and reverse transcriptase-polymerase chain reaction, respectively. The percentages of normal sperm motility and normal sperm morphology were significantly decreased in animals receiving METH, especially in escalating dose (ED METH) and escalating dose-binge (ED-binge METH) groups when compared with control. In addition, sperm concentrations in ED METH and ED-binge METH groups were numerically decreased. PR, ERα and ERβ immunoreactive cells were significantly decreased in spermatogonia, spermatogenic cells and especially in Sertoli cells in all METH-treated groups. Furthermore, messenger RNA expression of PR, ERα and ERβ were also significantly decreased in all METH-treated animals. These results indicate that METH can induce abnormal sperm quality. These changes of sperm quality may relate to the reduction of PR, ERα and ERβ expressions in male germ cells and Sertoli cells which are essential for spermatogenesis and development of sperm.

  11. OSBP-related protein 8 (ORP8) interacts with Homo sapiens sperm associated antigen 5 (SPAG5) and mediates oxysterol interference of HepG2 cell cycle

    SciTech Connect

    Zhong, Wenbin; Zhou, You; Li, Jiwei; Mysore, Raghavendra; Luo, Wei; Li, Shiqian; Chang, Mau-Sun; Olkkonen, Vesa M.; Yan, Daoguang

    2014-04-01

    We earlier identified OSBP-related protein 8 (ORP8) as an endoplasmic reticulum/nuclear envelope oxysterol-binding protein implicated in cellular lipid homeostasis, migration, and organization of the microtubule cytoskeleton. Here, a yeast two-hybrid screen identified Homo sapiens sperm associated antigen 5 (SPAG5)/Astrin as interaction partner of ORP8. The putative interaction was further confirmed by pull-down and co-immunoprecipitation assays. ORP8 did not colocalize with kinetochore-associated SPAG5 in mitotic HepG2 or HuH7 cells, but overexpressed ORP8 was capable of recruiting SPAG5 onto endoplasmic reticulum membranes in interphase cells. In our experiments, 25-hydroxycholesterol (25OHC) retarded the HepG2 cell cycle, causing accumulation in G2/M phase; ORP8 overexpression resulted in the same phenotype. Importantly, ORP8 knock-down dramatically inhibited the oxysterol effect on HepG2 cell cycle, suggesting a mediating role of ORP8. Furthermore, knock-down of SPAG5 significantly reduced the effects of both ORP8 overexpression and 25OHC on the cell cycle, placing SPAG5 downstream of the two cell-cycle interfering factors. Taken together, the present results suggest that ORP8 may via SPAG5 mediate oxysterol interference of the HepG2 cell cycle. - Highlights: • The oxysterol-binding protein ORP8 was found to interact with the mitotic regulator SPAG5/Astrin. • Treatment of HepG2 cells with 25-hydroxycholesterol caused cell cycle retardation in G2/M. • ORP8 overexpression caused a similar G2/M accumulation, and ORP8 knock-down reversed the 25-hydroxycholesterol effect. • Reduction of cellular of SPAG5/Astrin reversed the cell cycle effects of both 25-hydroxycholesterol and ORP8 overexpression. • Our results suggest that ORP8 mediates via SPAG5/Astrin the oxysterol interference of HepG2 cell cycle.

  12. Increased aneuploidy rate in sperm with fragmented DNA as determined by the sperm chromatin dispersion (SCD) test and FISH analysis.

    PubMed

    Muriel, Lourdes; Goyanes, Vicente; Segrelles, Enrique; Gosálvez, Jaime; Alvarez, Juan G; Fernández, José Luis

    2007-01-01

    Previous studies suggest that sperm DNA fragmentation may be associated with aneuploidy. However, currently available tests have not made it possible to simultaneously perform DNA fragmentation and chromosomal analyses on the same sperm cell. The recently introduced sperm chromatin dispersion (SCD) test allows users to determine this relationship. Semen samples from 16 males, including 4 fertile donors, 7 normozoospermic, 3 teratozoospermic, 1 asthenozoospermic, and 1 oligoasthenoteratozoospermic, were processed for DNA fragmentation analysis by the SCD test using the Halosperm kit. Three-color fluorescence in situ hybridization (FISH) was performed on SCD-processed slides to determine aneuploidy for chromosomes X, Y, and 18. Spermatozoa with DNA fragmentation showed a 4.4 +/- 1.9-fold increase in diploidy rate and a 5.9 +/- 3.5-fold increase in disomy rate compared to spermatozoa without DNA fragmentation. The overall aneuploidy rate was 4.6 +/- 2.0-fold higher in sperm with fragmented DNA (Wilcoxon rank test: P < .001 in the 3 comparisons). A higher frequency of DNA fragmentation was found in sperm cells containing sex chromosome aneuploidies originated in both first and second meiotic divisions. The observed increase in aneuploidy rate in sperm with fragmented DNA may suggest that the occurrence of aneuploidy during sperm maturation may lead to sperm DNA fragmentation as part of a genomic screening mechanism developed to genetically inactivate sperm with a defective genomic makeup.

  13. RNALocate: a resource for RNA subcellular localizations.

    PubMed

    Zhang, Ting; Tan, Puwen; Wang, Liqiang; Jin, Nana; Li, Yana; Zhang, Lin; Yang, Huan; Hu, Zhenyu; Zhang, Lining; Hu, Chunyu; Li, Chunhua; Qian, Kun; Zhang, Changjian; Huang, Yan; Li, Kongning; Lin, Hao; Wang, Dong

    2017-01-04

    Increasing evidence has revealed that RNA subcellular localization is a very important feature for deeply understanding RNA's biological functions after being transported into intra- or extra-cellular regions. RNALocate is a web-accessible database that aims to provide a high-quality RNA subcellular localization resource and facilitate future researches on RNA function or structure. The current version of RNALocate documents more than 37 700 manually curated RNA subcellular localization entries with experimental evidence, involving more than 21 800 RNAs with 42 subcellular localizations in 65 species, mainly including Homo sapiens, Mus musculus and Saccharomyces cerevisiae etc. Besides, RNA homology, sequence and interaction data have also been integrated into RNALocate. Users can access these data through online search, browse, blast and visualization tools. In conclusion, RNALocate will be of help in elucidating the entirety of RNA subcellular localization, and developing new prediction methods. The database is available at http://www.rna-society.org/rnalocate/.

  14. RNALocate: a resource for RNA subcellular localizations

    PubMed Central

    Zhang, Ting; Tan, Puwen; Wang, Liqiang; Jin, Nana; Li, Yana; Zhang, Lin; Yang, Huan; Hu, Zhenyu; Zhang, Lining; Hu, Chunyu; Li, Chunhua; Qian, Kun; Zhang, Changjian; Huang, Yan; Li, Kongning; Lin, Hao; Wang, Dong

    2017-01-01

    Increasing evidence has revealed that RNA subcellular localization is a very important feature for deeply understanding RNA's biological functions after being transported into intra- or extra-cellular regions. RNALocate is a web-accessible database that aims to provide a high-quality RNA subcellular localization resource and facilitate future researches on RNA function or structure. The current version of RNALocate documents more than 37 700 manually curated RNA subcellular localization entries with experimental evidence, involving more than 21 800 RNAs with 42 subcellular localizations in 65 species, mainly including Homo sapiens, Mus musculus and Saccharomyces cerevisiae etc. Besides, RNA homology, sequence and interaction data have also been integrated into RNALocate. Users can access these data through online search, browse, blast and visualization tools. In conclusion, RNALocate will be of help in elucidating the entirety of RNA subcellular localization, and developing new prediction methods. The database is available at http://www.rna-society.org/rnalocate/. PMID:27543076

  15. Subcellular storage compartments of bacteriopheophorbide sensitizers

    NASA Astrophysics Data System (ADS)

    Moser, Joerg G.; Dembeck, U.; Hubert, M.; Spengler, Bernhard; Bayer, Rainer; Wagner, Birgit

    1994-03-01

    Fluorescence colocalization with the Golgi specific stain, NBD-ceramide, and the mitochondrial localizing stain, Rhodamine 123, confirmed the earlier assumption that the Golgi apparatus is one of the prominent storage compartments for bacteriopheophorbide esters in OAT 75 SCLC cells and several amelanotic melanoma cell lines (A375, Melur SP18, SkAMel 25). Furthermore, a diffuse staining of mitochondria, of non-structured cytoplasm, and an additional storage in melanine vesicles of the amelanotic melanoma cells suggests further storage compartments with quantitatively different contributions to the phototoxicity of bacteriochlorophyll-derived photosensitizers. Independent observations of early phototoxic effects on microfilamentous networks, enzymatic activities (succinate dehydrogenase, lactate dehydrogenase), and redistribution phenomena following primary uptake of the sensitizers let us assume that only a part of the 108 molecules taken up by a cell contribute directly to phototoxicity. Thus it may be asked if a proper subcellular positioning of only a few sensitizer molecules may have similar phototoxic effects as the huge amounts stored at apparently ineffective sites.

  16. Metabolic rate limits the effect of sperm competition on mammalian spermatogenesis.

    PubMed

    delBarco-Trillo, Javier; Tourmente, Maximiliano; Roldan, Eduardo R S

    2013-01-01

    Sperm competition leads to increased sperm production in many taxa. This response may result from increases in testes size, changes in testicular architecture or changes in the kinetics of spermatogenesis, but the impact of each one of these processes on sperm production has not been studied in an integrated manner. Furthermore, such response may be limited in species with low mass-specific metabolic rate (MSMR), i.e., large-bodied species, because they cannot process energy and resources efficiently enough both at the organismic and cellular levels. Here we compare 99 mammalian species and show that higher levels of sperm competition correlated with a) higher proportions of seminiferous tubules, b) shorter seminiferous epithelium cycle lengths (SECL) which reduce the time required to produce sperm, and c) higher efficiencies of Sertoli cells (involved in sperm maturation). These responses to sperm competition, in turn, result in higher daily sperm production, more sperm stored in the epididymides, and more sperm in the ejaculate. However, the two processes that require processing resources at faster rates (SECL and efficiency of Sertoli cells) only respond to sperm competition in species with high MSMR. Thus, increases in sperm production with intense sperm competition occur via a complex network of mechanisms, but some are constrained by MSMR.

  17. Identification of the major metabolite of 2,5-bis(5-hydroxymethyl-2-thienyl)furan (NSC 652287), an antitumor agent, in the S9 subcellular fraction of dog liver cells.

    PubMed

    Phillip, Lawrence R; Jorden, Jean L; Rivera, Maria I; Wolfe, Tracy L; Upadhyayb, Kaye; Stinson, Sherman F

    2002-02-05

    Alpha-Terthienyl (1) is a trithiophene found widely distributed in plants. Other naturally occurring trithiophenes are less widely distributed, but nonetheless exhibit potent antiviral and cytotoxic activities. A synthetic analog of 1, 2,5-bis(5-hydroxymethyl-2-thienyl)furan (2; NSC 652287) has recently been shown to possess exceptional activity and selectivity against several cell lines of the National Cancer Institute (NCI) anticancer drug screen. When incubated with the S9 subcellular fraction of dog liver cells, the concentration of 2 was observed to decline as a function of time, with a concomitant increase in a significant, time-dependent concentration of an unknown entity. The results of electron-ionization mass spectrometric analysis of the metabolite indicate an increase in 14 amu over that of 2, leading to suspicions that either an oxidation or a methylation had occurred. Results of differential derivatization and accurate mass analysis allow us to propose that metabolism of 2 involves the biotransformation of one of the two hydroxymethyl groups of 2 into a carboxylic acid functionality. This is further supported by separate experiments involving chemical oxidation and S9 incubation of 5-[5-[5-hydroxymethyl-2-thienyl]-2-furanyl]-2-thiophenecarboxaldehyde: comparing the mass spectra and gas chromatographic retention times of the resulting products to those of the identified metabolite of 2 show all to be the same.

  18. Sperm number trumps sperm size in mammalian ejaculate evolution

    PubMed Central

    Fitzpatrick, John L.

    2015-01-01

    Postcopulatory sexual selection is widely accepted to underlie the extraordinary diversification of sperm morphology. However, why does it favour longer sperm in some taxa but shorter in others? Two recent hypotheses addressing this discrepancy offered contradictory explanations. Under the sperm dilution hypothesis, selection via sperm density in the female reproductive tract favours more but smaller sperm in large, but the reverse in small, species. Conversely, the metabolic constraint hypothesis maintains that ejaculates respond positively to selection in small endothermic animals with high metabolic rates, whereas low metabolic rates constrain their evolution in large species. Here, we resolve this debate by capitalizing on the substantial variation in mammalian body size and reproductive physiology. Evolutionary responses shifted from sperm length to number with increasing mammalian body size, thus supporting the sperm dilution hypothesis. Our findings demonstrate that body-size-mediated trade-offs between sperm size and number can explain the extreme diversification in sperm phenotypes. PMID:26582027

  19. Effect of sorafenib on sperm count and sperm motility in male Swiss albino mice

    PubMed Central

    Shetty, Surekha Devadasa; Bairy, Laxminarayana Kurady

    2015-01-01

    The issue of male germ line mutagenesis and the effects on developmental defects in the next generation has become increasingly high profile over recent years. Mutagenic substance affects germinal cells in the testis. Since the cells are undergoing different phases of cell division and maturation, it is an ideal system to study the effect of chemotherapeutic agents. There are lacunae in the literature on the effect of sorafenib on gonadal function. With background, a study was planned to evaluate the effects of sorafenib on sperm count and sperm motility in male Swiss albino mice. Male Swiss albino mice were used for the study. The animals were segregated into control, positive control (PC) and three treatment groups. PC received oral imatinib (100 mg/kg body weight) and treatment groups received 25, 50, and 100 mg/kg body weight of sorafenib orally for 7 consecutive days at intervals of 24 h between two administrations. The control group remained in the home cage for an equal duration of time to match their corresponding treatment groups. The animals were sacrificed at the end of 1st, 2nd, 4th, 5th, 7th, and 10th weeks after the last exposure to drug, respectively. Sperm suspensions were prepared and introduced into a counting chamber. Total sperm count and motility were recorded. There was a significant decrease in sperm count and sperm motility by sorafenib which was comparable with the effect of PC imatinib. Sorafenib adversely affects sperm count and sperm motility which are reversible after discontinuation of treatment. PMID:26605157

  20. Direct visualization of sperm competition and sperm storage in Drosophila.

    PubMed

    Civetta, A

    Drosophila females engage in multiple matings [1] [2] [3] [4] even though they can store sperm in specialized organs for most of their life [5]. The existence of sperm competition in Drosophila has been inferred from the proportion of progeny sired by the second male in double-mating experiments [6] [7] [8]. Investigators have used this approach to quantify genetic variation underlying sperm competition [8] [9] [10], to elucidate its genetic basis [11], to identify the dependence of different male competitive ability on the genotype of the females with which they mate [12] and to discern the potential role of sperm competition in species isolation [13] [14]. This approach assumes that the sperm from two males stored in a female compete to fertilize the eggs. The mechanism by which sperm competition is accomplished is still unknown, however. Here, fluorescence microscopy, cytometry, and differently labeled sperm were used to analyze the fate of sperm inside the female's sperm storage organs, to quantify sperm competition, and to assess how closely paternity success corresponds to the appearance and location of the sperm. The results show that the first male's sperm is retained for a shortened period if the female remates, and that the second males that sire more progeny either induce females to store and use more of their sperm or strongly displace resident sperm.

  1. Cholesterol Depletion Alters Cardiomyocyte Subcellular Signaling and Increases Contractility

    PubMed Central

    McIntosh, Victoria J.; Abou Samra, Abdul B.; Mohammad, Ramzi M.; Lasley, Robert D.

    2016-01-01

    Membrane cholesterol levels play an important factor in regulating cell function. Sarcolemmal cholesterol is concentrated in lipid rafts and caveolae, which are flask-shaped invaginations of the plasma membrane. The scaffolding protein caveolin permits the enrichment of cholesterol in caveolae, and caveolin interactions with numerous proteins regulate their function. The purpose of this study was to determine whether acute reductions in cardiomyocyte cholesterol levels alter subcellular protein kinase activation, intracellular Ca2+ and contractility. Methods: Ventricular myocytes, isolated from adult Sprague Dawley rats, were treated with the cholesterol reducing agent methyl-β-cyclodextrin (MβCD, 5 mM, 1 hr, room temperature). Total cellular cholesterol levels, caveolin-3 localization, subcellular, ERK and p38 mitogen activated protein kinase (MAPK) signaling, contractility, and [Ca2+]i were assessed. Results: Treatment with MβCD reduced cholesterol levels by ~45 and shifted caveolin-3 from cytoskeleton and triton-insoluble fractions to the triton-soluble fraction, and increased ERK isoform phosphorylation in cytoskeletal, cytosolic, triton-soluble and triton-insoluble membrane fractions without altering their subcellular distributions. In contrast the primary effect of MβCD was on p38 subcellular distribution of p38α with little effect on p38 phosphorylation. Cholesterol depletion increased cardiomyocyte twitch amplitude and the rates of shortening and relaxation in conjunction with increased diastolic and systolic [Ca2+]i. Conclusions: These results indicate that acute reductions in membrane cholesterol levels differentially modulate basal cardiomyocyte subcellular MAPK signaling, as well as increasing [Ca2+]i and contractility. PMID:27441649

  2. Triennial Reproduction Symposium: sperm characteristics that limit success of fertilization.

    PubMed

    Flowers, W L

    2013-07-01

    Current industry estimates of reproductive performance for cattle, sheep, and swine operations indicate that males contribute significantly to fertility failures. This appears to be due to the use of subfertile individuals and emphasizes the need for additional research in identifying characteristics of sperm that compromise fertilization. In theory, sperm characteristics, such as motility or the percentage of normal sperm, form a positive relationship with fertility that reaches a certain maximal fertility (i.e., an asymptotic relationship). It is clear that variation exists among males in terms of how fertility responds to increasing sperm dosage or numbers of normal sperm, both in the slope of the curve and the point at which the fertility reaches a maximum. Variations along the linear portion of fertility curves are due to compensable traits that are involved with the ability of sperm to penetrate the zona pellucida. It appears that most fertility curves reach their plateau when 70% of sperm possess a given compensable trait. The level of fertility at which the plateau occurs is determined by noncompensable traits that are associated with binding of sperm to the oolemma, syngamy, and subsequent development of the zygote. Several studies have shown differences in fertility among males that have similar levels of compensable traits but differed in their noncompensable characteristics. Compensable and noncompensable traits can estimate either individual or functional characteristics of sperm. Intuitively, functional traits such as in vitro penetration should provide a better indication of fertilization than individual ones such as motility. However, correlations of both types with fertility are very similar. Reasons for this may be related to how characteristics of sperm cells are influenced by the female reproductive tract after insemination. Sperm capacitation is a functional trait in boars that is quite different in vitro versus in vivo. If this relationship

  3. The regulation of spermatogenesis and sperm function in nematodes

    PubMed Central

    Ellis, Ronald E.; Stanfield, Gillian M.

    2014-01-01

    In the nematode C. elegans, both males and self-fertile hermaphrodites produce sperm. As a result, researchers have been able to use a broad range of genetic and genomic techniques to dissect all aspects of sperm development and function. Their results show that the early stages of spermatogenesis are controlled by transcriptional and translational processes, but later stages are dominated by protein kinases and phosphatases. Once spermatids are produced, they participate in many interactions with other cells — signals from the somatic gonad determine when sperm activate and begin to crawl, signals from the female reproductive tissues guide the sperm, and signals from sperm stimulate oocytes to mature and be ovulated. The sperm also show strong competitive interactions with other sperm and oocytes. Some of the molecules that mediate these processes have conserved functions in animal sperm, others are conserved proteins that have been adapted for new roles in nematode sperm, and some are novel proteins that provide insights into evolutionary change. The advent of new techniques should keep this system on the cutting edge of research in cellular and reproductive biology. PMID:24718317

  4. Intracytoplasmic Sperm Injection (ICSI)

    MedlinePlus

    ... to the inside of the egg (cytoplasm), where fertilization takes place. Sometimes the sperm cannot penetrate the ... ICSI) can be done along with in vitro fertilization (IVF) to help fertilize the egg. During ICSI, ...

  5. Low Sperm Count

    MedlinePlus

    ... unknown, it might be related to abnormal testicular temperature regulation. Varicoceles result in reduced quality of the ... can be permanently reduced. Overheating the testicles. Elevated temperatures impair sperm production and function. Although studies are ...

  6. Functional distribution of synapsin I in human sperm

    PubMed Central

    Coleman, William L.; Kulp, Adam C.; Venditti, Jennifer J.

    2015-01-01

    Proteins known to function during cell–cell communication and exocytosis in neurons and other secretory cells have recently been reported in human sperm. Synapsins are a group of proteins that have been very well characterized in neurons, but little is known about synapsin function in other cell types. Based upon previous findings and the known function of synapsin, we tested the hypothesis that synapsin I was present in human sperm. Washed, capacitated, and acrosome induced sperm preparations were used to evaluate the functional distribution of synapsin I using immunocytochemistry. Protein extracts from mouse brain, mouse testis/epididymis, and human semen were used for protein blotting techniques. Immunolocalization revealed synapsin I was enriched in the sperm equatorial segment. Protein extracts from mouse brain, mouse testis/epididymis, and human semen were positive for synapsin I using several different antibodies, and dot blot results were confirmed by Western blot analyses. Finally, treatment of capacitated and acrosome reaction induced samples with anti-synapsin antibodies significantly reduced sperm motility. Localization of synapsin I in human sperm is a novel finding. The association of synapsin I with the sperm equatorial segment and effects on motility are suggestive of a role associated with capacitation and/or acrosome reaction, processes that render sperm capable of fertilizing an oocyte. PMID:26566474

  7. Methamphetamine induces abnormal sperm morphology, low sperm concentration and apoptosis in the testis of male rats.

    PubMed

    Nudmamud-Thanoi, S; Thanoi, S

    2011-08-01

    Methamphetamine has been reported to be an important drug in the field of reproductive toxicology. The aim of this study was to investigate the effects of methamphetamine administrations on sperm morphology, sperm concentration and apoptotic activity inside seminiferous tubule in male rats. Rats were administered a dose of 8 mg kg(-1) , intraperitoneally (IP), for acute group and a dose of 4 mg kg(-1) , IP, once daily for 14 days for sub-acute group. Percentage of normal sperm morphology was decreased in acute group when compared with control. Total numbers of sperm count were significantly decreased in acute and sub-acute groups. Apoptotic activities were most abundant in the seminiferous tubules of acute treated animals with a highly significant increase in the number of apoptotic cells per tubule. Those effects of methamphetamine seem to be dose-dependent. The results suggest that methamphetamine not only works as drug of abuse in central nervous system, but also in gametogenesis of males.

  8. Formation of primary sperm conjugates in a haplogyne spider (Caponiidae, Araneae) with remarks on the evolution of sperm conjugation in spiders.

    PubMed

    Lipke, Elisabeth; Michalik, Peter

    2012-11-01

    Sperm conjugation, where two or more sperm are physically united, is a rare but widespread pheno-menon across the animal kingdom. One group well known for its different types of sperm conjugation are spiders. Particularly, haplogyne spiders show a high diversity of sperm traits. Besides individual cleistospermia, primary (synspermia) and secondary (coenospermia, "spermatophore") sperm conjugation occurs. However, the evolution of sperm conjugates and sperm is not understood in this group. Here, we look at how sperm are transferred in Caponiidae (Haplogynae) in pursuit of additional information about the evolution of sperm transfer forms in spiders. Additionally, we investigated the male reproductive system and spermatozoa using light- and transmission electron-microscopy and provide a 3D reconstruction of individual as of well as conjugated spermatozoa. Mature spermatozoa are characterized by an extremely elongated, helical nucleus resulting in the longest spider sperm known to date. At the end of spermiogenesis, synspermia are formed by complete fusion of four spermatids. Thus, synspermia might have evolved early within ecribellate Haplogynae. The fused sperm cells are surrounded by a prominent vesicular area. The function of the vesicular area remains still unknown but might be correlated with the capacitation process inside the female. Further phylogenetic and functional implications of the spermatozoa and sperm conjugation are discussed.

  9. Detection of nerve growth factor (NGF) and its specific receptor (TrkA) in ejaculated bovine sperm, and the effects of NGF on sperm function.

    PubMed

    Li, C; Sun, Y; Yi, K; Ma, Y; Sun, Y; Zhang, W; Zhou, X

    2010-12-01

    The objective was to confirm the presence of nerve growth factor (NGF) and its specific receptor, TrkA, in ejaculated bovine sperm, and to investigate the effects of NGF on specific aspects of bovine sperm function. Both TrkA transcripts and immunoreactivity typical of the translated protein were detected by RT-PCR and western blotting. However, only the NGF protein was detected in bovine sperm using western blotting, and there was no RT-PCR evidence for NGF transcripts in sperm. Using an immunofluorescent technique, NGF-immunoreactivity was localized to the sperm head and tail, whereas that of TrkA was detected in the acrosomal cap, nucleus, and tail regions When sperm were treated with exogenous NGF, both leptin secretion and sperm viability were increased (P < 0.05); moreover, the percentages of late apoptotic and dead sperm were increased (P < 0.05). However, NGF had no effects on insulin secretion, mitochondrial activity, intracellular calcium levels, or the acrosome reaction of sperm (P > 0.05). In conclusion, the presence of TrkA transcript, as well as NGF and TrkA immunoreactivity were confirmed in bovine sperm. Furthermore, exogenous NGF had significant effects on the secretion of leptin, cell viability, and sperm apoptosis. This study provided strong evidence that NGF/TrkA may have roles in regulation of sperm physiology and perhaps male fertility and infertility.

  10. Gadolinium, a mechano-sensitive channel blocker, inhibits osmosis-initiated motility of sea- and freshwater fish sperm, but does not affect human or ascidian sperm motility.

    PubMed

    Krasznai, Zoltán; Morisawa, Masaaki; Krasznai, Zoárd Tibor; Morisawa, Sachiko; Inaba, Kazuo; Bazsáné, Zsuzsa Kassai; Rubovszky, Bálint; Bodnár, Béla; Borsos, Antal; Márián, Teréz

    2003-08-01

    Exposure to hypo-osmotic or hyperosmotic environment triggers the initiation of fish sperm motility. In this article, we report that calcium and potassium channel blockers do not influence motility of puffer fish sperm but calmodulin antagonists reversibly decrease it, suggesting that calmodulin-Ca(2+) interactions are prerequisite for the initiation of sperm motility in this species. Gadolinium (a stretch activated ion channel blocker) decreased the motility of puffer fish sperm from 92 +/- 3% to 6 +/- 3% and that of carp sperm