399 SPERMATOZOA-MEDIATED GENE TRANSFER IS INFLUENCED BY SIZE, STRUCTURE, AND CONCENTRATION OF EXOGENOUS DNA

2007 ◽  
Vol 19 (1) ◽  
pp. 315
Author(s):  
W. B. Feitosa ◽  
M. P. Milazzotto ◽  
E. A. L. Martins ◽  
A. M. Rocha ◽  
J. L. Avanzo ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Gel Electrophoresis ◽  
Sperm Cell ◽  
Reference Data ◽  
Size Structure ◽  
Specific Protein ◽  
Sperm Cells ◽  
Exogenous Dna ◽  
Mammalian Sperm ◽  
Cell Association

Mammalian sperm cells have a spontaneous ability to take up exogenous DNA in a process regulated by specific protein. However, little is known about the effect of exogenous DNA on sperm cell association. The aim of this work was to evaluate the effect of size, concentration, and structure of exogenous DNA sequence in apoptosis and oncosis induction and the association with spermatozoa. To evaluate the effect of size and concentration of exogenous DNA, sequences of 2.2, 5.5, and 8.5 kb were used at the concentrations of 500 ng (Experiment 1) or 5 × 1011 molecules (Experiment 2). To assess the effect of structure (Experiment 3), 3 sequences of approximately 500 bp were used, containing 65.7% of AT (AT sequence), 46.4% of AT (IN sequence), and 38.6% of AT (GC sequence). To evaluate apoptosis and oncosis in all treatments, sperm cells were incubated with exogenous DNA for 1 h, stained with 2 µL of Yo-Pro (100 µM) for 20 min, and 10 µL of propidium iodide (6 µM) for 10 min, and then analyzed by flow cytometry. To evaluate the transfection rates, sperm cells, after incubation, were separated from non-associated exogenous DNA by 2.5% gel electrophoresis. DNA (genomic and exogenous) was extracted and association rates obtained by real-time PCR. Data were submitted to ANOVA and compared by Tukey's test (P ≤ 0.05). The number of viable, apoptotic, oncosis, or in initial apoptosis/late oncosis spermatozoa incubated with different concentrations or sequences of exogenous DNA did not differ among groups. In Experiment 1, a higher DNA association with sperm cells was observed in the 5.5 kb sequence in comparison with the 2.2 and 8.5 kb. In Experiment 2, the 8.5 kb sequence had the lower association with the spermatozoa in comparison with the 2.2 kb and 5.5 kb. In Experiment 3, there was a higher interaction of the AT sequence when compared to IN and GC sequences. Reference data suggest that big sequences of exogenous DNA have advantages in the interaction with spermatozoa. The present work is the first one to show that the advantage of the big sequences on interaction became a disadvantage in relation to the ability of these cells to internalize this DNA. Table 1. Quantification of different exogenous DNA associated to sperm cells Financial support was provided by FAPESP 03/10234-7 and 03/07456-8.

scholarly journals Formation of Native-like Mammalian Sperm Cell Chromatin with Folded Bull Protamine

2004 ◽  
Vol 279 (19) ◽  
pp. 20088-20095 ◽  
Author(s):  
Igor D. Vilfan ◽  
Christine C. Conwell ◽  
Nicholas V. Hud
Keyword(s):  
Disulfide Bonds ◽  
Sperm Cell ◽  
Sperm Chromatin ◽  
Sperm Cells ◽  
Dna Binding Domain ◽  
Mammalian Sperm ◽  
Intermolecular Disulfide Bonds ◽  
Bull Sperm ◽  
Terminal Domains

The DNA of most vertebrate sperm cells is packaged by protamines. The primary structure of mammalian protamine I can be divided into three domains, a central DNA binding domain that is arginine-rich and amino- and carboxyl-terminal domains that are rich in cysteine residues. In native bull sperm chromatin, intramolecular disulfide bonds hold the terminal domains of bull protamine folded back onto the central DNA binding domain, whereas intermolecular disulfide bonds between DNA-bound protamines help stabilize the chromatin of mature mammalian sperm cells. Folded bull protamine was used to condense DNAin vitrounder various solution conditions. Using transmission electron microscopy and light scattering, we show that bull protamine forms particles with DNA that are morphologically similar to the subunits of native bull sperm chromatin. In addition, the stability provided by intermolecular disulfide bonds formed between bull protamine molecules withinin vitroDNA condensates is comparable with that observed for native bull sperm chromatin. The importance of the bull protamine terminal domains in controlling the bull sperm chromatin morphology is indicated by our observation that DNA condensates formed under identical conditions with a fish protamine, which lacks cysteine-rich terminal domains, do not produce as uniform structures as bull protamine. A model is also presented for the bull protamine·DNA complex in native sperm cell chromatin that provides an explanation for the positions of the cysteine residues in bull protamine that form intermolecular disulfide bonds.

Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells

1993 ◽  
Vol 34 (2) ◽  
pp. 133-139 ◽  
Author(s):  
Maura Francolini ◽  
Marialuisa Lavitrano ◽  
Carla Lora Lamia ◽  
Deborah French ◽  
Luigi Frati ◽  
...  
Keyword(s):  
Sperm Cells ◽  
Exogenous Dna ◽  
Mammalian Sperm

scholarly journals Persistent directional growth capability in Arabidopsis thaliana pollen tubes after nuclear elimination from the apex

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kazuki Motomura ◽  
Hidenori Takeuchi ◽  
Michitaka Notaguchi ◽  
Haruna Tsuchi ◽  
Atsushi Takeda ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Pollen Tube ◽  
Sperm Cell ◽  
Pollen Tubes ◽  
Vegetative Nucleus ◽  
Sperm Cells ◽  
Apical Region ◽  
Basal Region ◽  
Specific Expression ◽  
Directional Growth

AbstractDuring the double fertilization process, pollen tubes deliver two sperm cells to an ovule containing the female gametes. In the pollen tube, the vegetative nucleus and sperm cells move together to the apical region where the vegetative nucleus is thought to play a crucial role in controlling the direction and growth of the pollen tube. Here, we report the generation of pollen tubes in Arabidopsis thaliana whose vegetative nucleus and sperm cells are isolated and sealed by callose plugs in the basal region due to apical transport defects induced by mutations in the WPP domain-interacting tail-anchored proteins (WITs) and sperm cell-specific expression of a dominant mutant of the CALLOSE SYNTHASE 3 protein. Through pollen-tube guidance assays, we show that the physiologically anuclear mutant pollen tubes maintain the ability to grow and enter ovules. Our findings provide insight into the sperm cell delivery mechanism and illustrate the independence of the tip-localized vegetative nucleus from directional growth control of the pollen tube.

Distribution, expression and germ line transmission of exogenous DNA sequences following microinjection into Xenopus laevis eggs

Development ◽  
1987 ◽  
Vol 99 (1) ◽  
pp. 15-23
Author(s):  
L.D. Etkin ◽  
B. Pearman
Keyword(s):  
Xenopus Laevis ◽  
Dna Sequences ◽  
Copy Number ◽  
Germ Line ◽  
Gene Promoter ◽  
Early Gene ◽  
Mosaic Pattern ◽  
Exogenous Dna ◽  
Gene Coding ◽  
Germ Line Transmission

We analysed the fate, expression and germ line transmission of exogenous DNA which was microinjected into fertilized eggs of Xenopus laevis. DNA was injected into fertilized eggs within 1 h following fertilization. The injected DNA was dispersed around the site of injection and became localized to cleavage nuclei by stage 6. Injected DNA persisted in the tissues of 6- to 8-month-old frogs and exhibited a mosaic pattern of distribution with regard to the presence or absence and copy number between different tissues. We detected the exogenous DNA sequences in 60% of injected frogs. Restriction digestion analysis of this DNA suggested that it is not rearranged and was organized as head-to-tail multimers. The copy number varied from 2 to 30 copies/cell in various tissues of the same frog. Plasmid pSV2CAT which contains the prokaryotic gene coding for chloramphenicol acetyl transferase (CAT) enzyme linked to the SV40 early gene promoter was expressed in 50% of the animals containing the gene. The pattern of expression, however, varied between different animals and could not be correlated with copy number. We also showed that the exogenous DNA sequences were transmitted through the male germ line and that each offspring contained the gene integrated into a different region of the genome.

scholarly journals Influence of Campylobacter fetus subsp. fetus on ram sperm cell quality

2008 ◽  
Vol 57 (11) ◽  
pp. 1405-1410 ◽  
Author(s):  
Tidhar Zan Bar ◽  
Ronen Yehuda ◽  
Tomer Hacham ◽  
Sigal Krupnik ◽  
Benjamin Bartoov
Keyword(s):  
Sperm Cell ◽  
Sperm Cells ◽  
Male Factor ◽  
Motility Index ◽  
Fas Receptor ◽  
Campylobacter Fetus ◽  
Fetus Subsp ◽  
Ram Sperm ◽  
Female Sheep

Campylobacter fetus subsp. fetus infection can occur in female sheep, causing infertility or abortion. Despite extensive research on the effect of these bacteria on female fertility, little research has been done on the influence of C. fetus subsp. fetus on the male factor. Our objective was to examine the influence of C. fetus subsp. fetus on ram sperm. Motility index, percentage of live spermatozoa, mean αt value (an indication of the chromatin stability of the sperm cell) and percentage of sperm cells expressing the FAS receptor were measured in sperm incubated in the presence or absence of C. fetus subsp. fetus. The motility index and viability of sperm incubated with the bacteria were lower than those of untreated sperm samples after 5 h. In bacteria-incubated sperm cells, the percentage expressing FAS receptor was already significantly elevated at 15 min. Bacteria-incubated sperm showed a greater prevalence of morphological damage. The bacteria were attached to tail and acrosome regions, and the sperm damage was concentrated in both the motility and chromatin regions. Bacteria-infected sperm cells showed a decrease in motility, increase in early acrosome reaction and chromatin damage. Similar effects were induced by incubation of the sperm with supernatants from C. fetus subsp. fetus cultures. Thus this study demonstrates that C. fetus subsp. fetus has a detrimental effect on the quality of ram sperm.

scholarly journals Surface glycoproteins of human spermatozoa

1986 ◽  
Vol 82 (1) ◽  
pp. 11-22
Author(s):  
M. Kallajoki ◽  
I. Virtanen ◽  
J. Suominen
Keyword(s):  
Plasma Membrane ◽  
Gel Electrophoresis ◽  
Soluble Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Human Spermatozoa ◽  
Galactose Oxidase ◽  
Sperm Cells ◽  
Neuraminidase Treatment ◽  
Triton X

The surface membrane glycoprotein composition of human spermatozoa has been studied by introducing radioactive label into galactosyl (Gal) and N-acetylgalactosaminyl (GalNAc) residues by using the galactose oxidase/NaB3H4 method. Triton X-100 extracts and Triton X-100-resistant cytoskeletal residues were subjected to analysis by polyacrylamide gel electrophoresis. The distribution of the radiolabel in sperm cells was studied by light-microscopic auto-radiography. The grains were evenly distributed on the cells by the labelling methods used. The Triton X-100 treatment did not affect sperm morphology at the light-microscopic level, but in transmission electron microscopy the plasma membrane covering the acrosome was removed totally, together with most of the acrosomal membranes and acrosomal contents. Plasma membrane residues were, however, always found in the postacrosomal region. Borohydride alone without oxidative pretreatment labelled two polypeptides of molecular weights (Mr) 48,000 and 43,000 in the Triton X-100-soluble fraction. When the Gal/GalNAc residues were labelled by galactose oxidase pretreatment 120,000, 105,000, 78,000 and 68,000 Mr glycoproteins were revealed. When additional neuraminidase treatment was used to remove terminal sialic acid residues, the total labelling intensity was increased two- to fivefold and additional 36,000 and 20,000 Mr glycoproteins were revealed. The Triton X-100-resistant cytoskeletal residue contained 53–75% of the total radioactivity bound in sperm cells. When these components were analysed by polyacrylamide gel electrophoresis, all the major bands found in the Triton X-100-soluble fraction were detected and also some radioactivity was incorporated into the major bands visualized by protein staining. In the present study we describe several human sperm glycoproteins, which seem to be distributed evenly on the sperm cells. Detergent extraction, producing cytoskeletal models, appeared to leave most of the glycoproteins detectable in the extraction residues also with the apparent enrichment of a single 68,000 Mr glycoprotein.

scholarly journals A Study of Aflatoxin on Sperm Cell Quality for Elderly People

2018 ◽  
Vol 6 (2) ◽  
pp. 45-49
Author(s):  
Akila CR ◽  
Dinesh Babu J ◽  
Sravan Kumar P ◽  
Vinaya B
Keyword(s):  
Elderly People ◽  
Sperm Cell ◽  
Sperm Cells

Mycotoxins represent poisonous materials produced via certain types of growths. Among different mycotoxins, aflatoxins are viewed as plainly hazardous, taking into account that they are portrayed as cancer-causing for creatures and individuals. The admission of aflatoxins by means of feeds or nourishments should cause pernicious results on creatures' or people's wellbeing. Exploration on creatures has indicated that the overall casing circumstance just as a portion of the blood boundaries, particularly those of the liver may be adversely influenced with aflatoxin the executives. As to conceptive device, in spite of the fact that not remarkably examined, a few specialists bolster the helpless impacts of aflatoxins both on women or on grown-up guys. More precisely, in male, the scale and weight of the genital organs, the spermatogenesis, the number, the motility and the morphology of sperm cells just as hormones' fixations can be influenced after presentation of the creatures to aflatoxins, making barrenness inconveniences additional normal. Most examination allude to lab and significantly less to viable creatures, while least complex two investigations look for counsel from the doable issues of barrenness on men in view of aflatoxins. Since imitation is made one out of the greatest fundamental areas of creature cultivation, exceptional intrigue should be paid to supplements with the goal that the chance of the aflatoxin admission by creatures could be evacuated, the creature wellbeing extraordinarily in regards to the conceptive gadget may be covered and financial misfortunes could be enhanced.

scholarly journals Expression and function of ras proto-oncogene proteins in human sperm cells

1992 ◽  
Vol 102 (3) ◽  
pp. 487-494 ◽  
Author(s):  
R.K. Naz ◽  
K. Ahmad ◽  
P. Kaplan
Keyword(s):  
Sperm Cell ◽  
Acrosome Reaction ◽  
Cell Function ◽  
Beat Frequency ◽  
Human Sperm ◽  
Sperm Cells ◽  
Ras Protein ◽  
Head Displacement ◽  
Sperm Penetration ◽  
And Function

The presence and role of c-ras proteins were investigated in mature human sperm cells. The v-H-ras monoclonal antibody (mAb) against the c-ras protein, p21, reacted specifically with the acrosomal region of methanol-fixed as well as unfixed-live capacitated and non-capacitated human sperm cell in the indirect immunofluorescence technique. The v-H-ras mAb predominantly recognized c-ras protein of 21 kDa on the Western blot of lithium diiodosalicylate (LIS)-solubilized human sperm preparation. The incubation of sperm cells with v-H-ras mAb affected the sperm cell function in the human sperm penetration assay. The antibody significantly reduced the acrosome reaction and release of acrosin activity from the sperm cells. There was no effect of the mAb on percentage motility, although the mAb significantly affected various motility characteristics such as linearity, amplitude of lateral head displacement (ALH) and beat frequency, the motility parameters involved in the hyperactivation phenomenon of sperm cells leading to capacitation and acrosome reaction. These results suggest that the c-ras or c-ras-like proteins are present in mature sperm cell and may have a role in capacitation and/or acrosome reaction of human sperm cell.

scholarly journals Direct introduction of cloned DNA into the sea urchin zygote nucleus, and fate of injected DNA

Development ◽  
1988 ◽  
Vol 102 (2) ◽  
pp. 287-299 ◽  
Author(s):  
R.R. Franks ◽  
B.R. Hough-Evans ◽  
R.J. Britten ◽  
E.H. Davidson
Keyword(s):  
Dna Sequences ◽  
Larval Growth ◽  
Exogenous Dna ◽  
Zygote Nucleus ◽  
Cell Lineages ◽  
Larval Stages ◽  
Injection Process ◽  
End To End ◽  
Nuclear Injection ◽  
Cytoplasmic Injection

A method is described for microinjection of cloned DNA into the zygote nucleus of Lytechinus variegatus. Eggs of this species are unusually transparent, facilitating visual monitoring of the injection process. The initial fate of injected DNA fragments appears similar to that observed earlier for exogenous DNA injected into unfertilized egg cytoplasm. Thus after end-to-end ligation, it is replicated after a lag of several hours to an extent indicating that it probably participates in most of the later rounds of DNA synthesis undergone by the host cell genomes during cleavage. The different consequences of nuclear versus cytoplasmic injection are evident at advanced larval stages. Larvae descendant from eggs in which exogenous DNA was injected into the nuclei are four times more likely (32% versus 8%) to retain this DNA in cell lineages that replicate very extensively during larval growth, i.e. the lineages contributing to the imaginal rudiment, and thus to display greatly enhanced contents of the exogenous DNA. Similarly, 36% of postmetamorphic juveniles from a nuclear injection sample retained the exogenous DNA sequences, compared to 12% of juveniles from a cytoplasmic injection sample. However, the number of copies of the exogenous DNA sequences retained per average genome in postmetamorphic juveniles was usually less than 0.1 (range 0.05-50), and genome blot hybridizations indicate that these sequences are organized as integrated, randomly oriented, end-to-end molecular concatenates. It follows that only a small fraction of the cells of the average juvenile usually retains the exogenous sequences. Thus, even when introduced by nuclear microinjection, the stable incorporation of exogenous DNA in the embryo occurs in a mosaic fashion, although in many recipients the DNA enters a wider range of cell lineages than is typical after cytoplasmic injection. Nuclear injection would probably be the route of choice for studies of exogenous DNA function in the postembryonic larval rudiment.

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