scholarly journals AN ANALYSIS OF THE INACTIVATION OF THE FROG SPERM NUCLEUS BY TOLUIDINE BLUE

1952 ◽  
Vol 35 (5) ◽  
pp. 761-780 ◽  
Author(s):  
Robert Briggs
Keyword(s):  
Toluidine Blue ◽  
Sperm Nucleus ◽  
Sperm Cells ◽  
Dye Molecules ◽  
Ph Measurements ◽  
Complete Inactivation ◽  
Minimal Quantity ◽  
Sperm Nuclei ◽  
Sperm Chromosomes ◽  
Ph Range

Toluidine blue, applied to frog sperm under appropriate conditions, inactivates specifically the sperm nucleus, leaving the extranuclear parts of the cell undamaged. Thus, the dye-treated spermatozoa stimulate eggs to cleave normally, but contribute no chromosomes to the resulting embryos, which develop as typical gynogenetic haploids. The concentration of dye required to produce this inactivation varies with pH. Measurements made over the entire pH range which can be tolerated by sperm cells showed that in the lower part of the range (5 to 7) the effective dye concentration was about 5 x 10–6 M; in the intermediate range (7 to 8.5) it was 1 x –6 to 1 x –7 M; and for the higher pH values (8.5 to 10.0) it was about 5 x –8 M. Using sperm suspensions containing 1500 cells per c. mm. these concentrations of dye produced specific inactivation of the sperm nuclei within 7 to 60 minutes at 18°C. Tests of the reversibility of the inactivation were made by transferring the sperm from the dye to a dilute Ringer's solution after a known degree of inactivation had been produced. Following removal of the dye the sperm cells were tested on eggs over a period of 2 hours. During this time there was no indication of a reversal of the inactivation. Microscopic observations of sperm treated with –5 M or 5 X –5 M dye show that the dye is taken up by the sperm nucleus, which is faintly but definitely stained. The dye appears to be uniformly distributed in the nucleus, while extranuclear structures remain unstained. Measurements of the amount of dye bound per sperm nucleus indicate that the minimal quantity required for complete inactivation is about 6.7 x –18 mole, while the maximal amount which can be bound without injury to extranuclear structures is about 1.5 x –16 mole. The value obtained for the minimal requirement (6.7 X 16 mole = 4 X 6 molecules) suggests that there are roughly 4 million binding sites in the nucleus which, when blocked by dye molecules, somehow prevent the sperm chromosomes from participating in the development of the egg.

A High Resolution Cytochemical Study of Nuclear ATPase in Human Spermatozoa

1975 ◽  
Vol 33 ◽  
pp. 594-595
Author(s):  
A. Sosa ◽  
L. Calzada
Keyword(s):  
High Resolution ◽  
Atpase Activity ◽  
Nuclear Membrane ◽  
Human Sperm ◽  
Sperm Nucleus ◽  
Human Spermatozoa ◽  
Cytochemical Study ◽  
Membrane Bound ◽  
Sperm Nuclei ◽  
Interchromatin Space

The dependence of nuclear metabolism on the function of the nuclear membrane is not well understood. Whether or not the function of the nuclear membrane is partial or totally responsible of the repressed template activity of human sperm nucleus has not at present been elucidated. One of the membrane-bound enzymatic activities which is concerned with the mechanisms whereby substances are thought to cross cell membranes is adenosintriphosphatase (ATPase). This prompted its characterization and distribution by high resolution photogrammetry on isolated human sperm nuclei. Isolated human spermatozoa nuclei were obtained as previously described. ATPase activity was demonstrated by the method of Wachstein and Meisel modified by Marchesi and Palade. ATPase activity was identified as dense and irregularly distributed granules confined to the internal leaflet of the nuclear membrane. Within the nucleus the appearance of the reaction product occurs as homogenous and dense precipitates in the interchromatin space.

Role of karyoplasm in the emergence of capacity of egg cytoplasm to induce DNA synthesis in transplanted sperm nuclei

Development ◽  
1976 ◽  
Vol 36 (1) ◽  
pp. 67-72
Author(s):  
M. N. Skoblina
Keyword(s):  
Dna Synthesis ◽  
Sperm Nucleus ◽  
Germinal Vesicles ◽  
Sperm Nuclei

The behaviour of sperm nuclei was studied both in the cytoplasm of intact toad oocytes undergoing maturation and the cytoplasm of oocytes matured without germinal vesicles. The behaviour of the nuclei of pronase-treated sperm injected in the mature egg cytoplasm was shown to be exactly similar to that of the sperm nucleus after fertilization, i.e. they swelled, synthesized DNA, and divided. No changes in such sperm nuclei could be detected in the cytoplasm of the oocytes matured without germinal vesicles.

scholarly journals Changes in the Level of DNA Fragmentation in Sperm Cells detected by Acridine Orange Test in Men with Sub/infertility Treated with Nutritional Supplement PAPA

Folia Medica ◽  
2020 ◽  
Vol 62 (1) ◽  
pp. 112-116
Author(s):  
Stoil N. Tomov ◽  
Radoslava St. Stoyanova ◽  
Pepa K. Atanasova ◽  
Ivan Y. Dechev
Keyword(s):  
Acridine Orange ◽  
Dna Fragmentation ◽  
Physiological Condition ◽  
Nutritional Supplement ◽  
Sperm Cells ◽  
Sperm Dna Fragmentation ◽  
Double Stranded Dna ◽  
Sperm Dna ◽  
Before And After ◽  
Sperm Nuclei

Background: When diagnosing and treating male infertility it is important to determine whether there are defects in the maturation process of sperm nuclei. Using nutritional supplements can improve the morphological and physiological condition of the spermatozoa. In recent years there has been an increase in the usage of supplements with different compositions which strives to determine the best combination and avoid side effects. &nbsp; Aim: To study the effect of PAPA nutritional supplement on the levels of DNA fragmentation of sperm cells tested with acridine orange test (single stranded DNA against double stranded DNA) in men with sub/infertility. &nbsp; Materials and methods: 48 men with confirmed sub/infertility underwent treatment for three months with nutritional supplement PAPA containing 9 micronutrients. The differences in levels of DNA fragmentation were determined with acridine orange test, which was conducted before and after the treatment. &nbsp; Results: The results were statistically significant (p<0.001) showing an increase in the number of green spermatozoa (normal DNA), and a decrease of damaged ones (orange and red). After treatment the level of sperm DNA fragmentation decreased by 10.2%. &nbsp; Conclusion: Men with confirmed sub/infertility that took nutritional supplement PAPA for three moths showed a decrease in DNA fragmentation levels of 10.2% determined by AO test which implies an improvement of male fertility levels.

Coiling of the Flagellar Shaft in the Head Region of the Mature Sperm of Nucella (Purpura) Lapillus (L.)

1969 ◽  
Vol 5 (1) ◽  
pp. 211-225
Author(s):  
MURIEL WALKER
Keyword(s):  
Sea Water ◽  
Nuclear Material ◽  
Sperm Nucleus ◽  
Mature Sperm ◽  
Langmuir Trough ◽  
Head Region ◽  
Anterior Portion ◽  
Normal Concentration ◽  
Ph Range ◽  
Dry Out

The sperm of Nucella are long and threadlike. The flagellar shaft runs from the acrosome at the front of the head to the tip of the tail. Its anterior portion, the head shaft, is ensheathed by the nucleus. If a suspension of sperm in normal-concentration sea water is observed under phase contrast, and allowed to dry out slowly, the nuclear material of sperm near the edge of the coverglass swells and the head shafts of these sperm are thrown into gentle spirals within the nuclei. In some sperm the nuclear material disperses completely, and the front threequarters of the head shaft springs into a tight right-handed coil of 5-7 turns. Instantaneous coiling of the head shaft may also be induced by treatment of the sperm with x 2 concentrated sea water or 0.01 % (w/v) solution of sodium lauryl sulphate in sea water. The enzymes pronase and trypsin at a concentration of 50µg/ml in sea water at pH 8.0 cause dispersion of the head nucleoprotein and subsequently the head shaft forms a loose coil. The appearance and activity of the sperm do not change perceptibly over a pH range of 5.5-8.5. Sperm spread on a Langmuir trough containing normal concentration sea water were negatively stained with phosphotungstic acid and examined with an electron microscope. After such treatment the nuclei are partially spread and the fibrils of the head shafts appear twisted as the wires of an electrical flex. The twisted flagellar fibrils are seen more clearly in negatively stained head shafts of sperm whose nuclei have been completely dispersed by spreading on a Langmuir trough containing x2 concentrated sea water. Negatively stained preparations of sperm treated with enzymes show sperm with twisted and coiled head shafts. The nucleoprotein of these sperm is disaggregated into strips or sheets, or may appear as a mass of branching fibres. The flagellar fibres of the enzyme-treated sperm are often bent or broken. Longitudinal sections of mature sperm heads show that in the intact sperm the fibres of the head shaft are not twisted but run straight throughout the length of the head. Sections from the testis and testicular duct show that microtubules are present in sperm in the testis but absent from mature sperm in the testicular duct. It is suggested that the head shaft, as a consequence of some event in spermiogenesis, has an inherent tendency to twist and coil but in the mature sperm it is ‘strait-jacketed’ by the sperm nucleus.

Mechanisms of displacement of sperm basic nuclear proteins in mammals. An in vitro simulation of post-fertilization results

1982 ◽  
Vol 53 (1) ◽  
pp. 227-244
Author(s):  
T.C. Rodman ◽  
F.H. Pruslin ◽  
V.G. Allfrey
Keyword(s):  
Sperm Nucleus ◽  
Nuclear Proteins ◽  
Mouse Sperm ◽  
Basic Proteins ◽  
Molecular Events ◽  
In Vitro Simulation ◽  
Sperm Nuclei ◽  
Two Parameters

A standardized cytological preparation of mature mouse sperm has been devised to serve as an in vitro system for probing the intra-ooplasmic molecular events of transformation of the fertilizing sperm. Two parameters of the early phase of transformation in vivo are defined at the resolution of the light microscope: deletion of sperm-unique nuclear proteins, detectable by immunofluorescence, and retention of homogeneity of the residual DNA complex, with intact chromatin boundaries detectable by ethidium bromide staining. These studies show that both parameters are conserved when in vitro sperm preparations are treated with NaCl under reducing conditions. The deletion of 2 different classes of the unique basic proteins of mouse sperm nuclei is specified by the NaCl concentration: 0.7 M-NaCl displaces the non-protamine class but not the protamines, while 1 M-NaCl displaces both. On the other hand the effects of treatment with trypsin at various concentrations and intervals are less consistent with the in vivo parameters, indicating fragmentation and displacement, not only of the sperm-unique basic proteins, but also of structural proteins believed to maintain the fundamental cohesive organization of the DNA matrix. These observations suggest that mechanisms other than proteolysis, e.g. localized changes in ionic concentrations, may participate in the post-fertilization displacement of the sperm-unique nuclear proteins in vivo. This study also supports the validity of the in vitro simulation as a model with which to probe the progression of transformation of the sperm nucleus to the zygote pronucleus.

Insemination of the archegonium and fertilization in Taxus baccata L

1988 ◽  
Vol 89 (4) ◽  
pp. 551-560
Author(s):  
ROGER I. PENNELL ◽  
PETER R. BELL
Keyword(s):  
Pollen Tube ◽  
Male Gametophyte ◽  
Sperm Nucleus ◽  
Taxus Baccata ◽  
Close Apposition ◽  
Cell Cytoplasm ◽  
Spermatogenous Cell ◽  
Tube Lumen ◽  
Sperm Nuclei ◽  
Progressive Enlargement

A study of fertilization in Taxus baccata in the electron microscope has revealed novel features. Insemination of the archegonium is facilitated by local perforation of the wall of the young pollen tube. Digestion of the wall begins before the pollen tube pierces the megaspore membrane but is not completed until its tip makes contact with the neck cells of the archegonium. As soon as a pore is formed a single sperm nucleus and some cytoplasm of the male gametophyte enter the archegonium. Which of the paired sperm nuclei move from the pollen tube into the archegonium appears to be a matter of chance. Close apposition of sperm nucleus and egg nucleus is followed by the formation of numerous points of contact between the two. The membranes fuse at these points and pores are rapidly formed. The progressive enlargement of these pores ultimately eliminates any partitions and yields the zygotic nucleus. There is a possibility that, as in some other gymnosperms, the plastids and mitochondria of the zygote come in part from the male gametophyte, but whether from the remains of the spermatogenous cell cytoplasm or from the. pollen tube lumen is not clear.

scholarly journals Reduction of the incidence of polyspermic penetration into porcine oocytes by pretreatment of fresh spermatozoa with adenosine and a transient co-incubation of the gametes with caffeine

Reproduction ◽  
2004 ◽  
Vol 128 (6) ◽  
pp. 789-800 ◽  
Author(s):  
Hiroaki Funahashi ◽  
Raquel Romar
Keyword(s):  
Incubation Period ◽  
Culture Period ◽  
Sperm Cells ◽  
Free Medium ◽  
Exposure Treatment ◽  
Pretreatment Effect ◽  
The Mean ◽  
Sperm Nuclei

To reduce the incidence of polyspermic penetration, the effects of transient exposure of washed fresh spermatozoa to caffeine in a brief co-culture in vitro fertilization (IVF) system were examined. A pretreatment effect of spermatozoa with adenosine was also examined. When 5 mmol caffeine/l was supplemented during periods of co-culture and additional culture periods until 8 h after insemination, a shortened co-incubation period of gametes (30 denuded oocytes in 100 μl modified Medium 199-suspended spermatozoa at 2.5 ×105 sperm/ml) from 30 to 5 min increased the monospermy rate in total mature oocytes examined. The number of spermatozoa binding to the zona surface was significantly lower in oocytes co-cultured for 5 min (33.1 ± 2.2) than 8 h (207.6 ± 13.7). A limited exposure of gametes to 5 mmol caffeine/l only during a transient co-culture period for 5 or 30 min significantly reduced the mean number of sperm cells that penetrated into the oocyte. Transient exposure of spermatozoa to caffeine for only 5 min increased the percentage of capacitated cells but not acrosome-reacted cells, as compared with a whole exposure treatment. Furthermore, preincubation of spermatozoa with 10 μmol adenosine/l for 90 min increased both the incidence of capacitated cells and the monospermy rate and consequently decreased the number of sperm cells that penetrated into the oocyte. In conclusion, these results have demonstrated that a new transient co-incubation IVF system, in which denuded oocytes are co-cultured with spermatozoa in medium containing caffeine for 5 to 30 min and then continuing the culture in caffeine-free medium, will reduce the incidence of polyspermic penetration. Preincubation of fresh spermatozoa with adenosine before the transient co-incubation IVF can also improve the monospermy rate. Furthermore, asynchrony in the morphology of sperm nuclei in polyspermic oocytes was reduced by the pretreatment with adenosine and a brief exposure to caffeine.

scholarly journals Analysis of the Bacterial Sulphur System

2013 ◽  
Vol 825 ◽  
pp. 190-193
Author(s):  
Rachel M. Candy ◽  
Kyle R. Blight ◽  
David E. Ralph
Keyword(s):  
Bacterial Cell ◽  
Mine Drainage ◽  
Solid Phase ◽  
Low Grade ◽  
Ph Measurements ◽  
Batch Cultures ◽  
Partial Pressures ◽  
S Oxidation ◽  
Ph Range

Heterogeneous bacterial sulphur systems are inherently complicated. However, developing an understanding of the influence of environmental factors such as pH,Iand PCO2is important for a number of fields. Examples of these include minimising acid mine drainage and maximising metal recovery from low-grade sulphide minerals. Measuring the effect of these factors on the extent and rate of sulphur (S) oxidation is complicated by the presence and nature of solid phase elemental S. The rate and extent of S oxidation can be determined indirectly via the reaction product, H2SO4, which was quantified using pH measurements in this study. The method was critically dependent on the quality of pH data but proved effective in providing rate constants for the catalysed S oxidation reaction and yield (biomass/substrate) estimates in the range pH > 1.5. IncreasingIover the range 0.176 0.367 mol L-1decreased bacterial cell yields but increased the rate of sulphur oxidation significantly. Partial pressures of CO2in the range of 0.039 1.18% v/v produced no significant effect on the rates of S oxidation or bacterial cell yields. Bacterial cell yields were not affected in the pH range 1.5 2.5, however the rate of S oxidation increased significantly from pH 2.0 2.5. In the range pH < 1.5 the batch cultures progressed and although no reliable rate data was recorded cell yields decreased from 7.43 to 2.05 (× 1012cells mol-1) at pH 1.5 to 1.0 respectively.

scholarly journals Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse.

1986 ◽  
Vol 102 (3) ◽  
pp. 1039-1046 ◽  
Author(s):  
H J Clarke ◽  
Y Masui
Keyword(s):  
Morphological Changes ◽  
Small Mass ◽  
Sperm Nucleus ◽  
Metaphase Chromosomes ◽  
Oocyte Meiosis ◽  
Maturation In Vitro ◽  
Sperm Penetration ◽  
Sperm Nuclei ◽  
Metaphase I

Zona-free oocytes of the mouse were inseminated at prometaphase I or metaphase I of meiotic maturation in vitro, and the behavior of the sperm nuclei within the oocyte cytoplasm was examined. If the oocytes were penetrated by up to three sperm, maturation continued during subsequent incubation and became arrested at metaphase II. Meanwhile, each sperm nucleus underwent the following changes. First, the chromatin became slightly dispersed. By 6 h after insemination, this dispersed chromatin had become coalesced into a small mass, from which short chromosomal arms later became projected. Between 12 and 18 h after insemination, each mass of chromatin became resolved into 20 discrete metaphase chromosomes. In contrast, if oocytes were penetrated by four to six sperm, oocyte meiosis was arrested at metaphase I, and each sperm nucleus was transformed into a small mass of chromatin rather than into metaphase chromosomes. If oocytes were penetrated by more than six sperm, the maternal chromosomes became either decondensed or pycnotic, and the sperm nuclei were transformed into larger masses of chromatin. As control experiments, immature and fully mature metaphase II oocytes were inseminated. In the immature oocytes, which were kept immature by exposure to dibutyryl cyclic AMP, no morphological changes in the sperm nucleus were observed. On the other hand, in the fully mature oocytes, which were activated by sperm penetration, the sperm nucleus was transformed into the male pronucleus. Therefore, the cytoplasm of the maturing oocyte develops an activity that can transform the highly condensed chromatin of the sperm into metaphase chromosomes. However, the capacity of an oocyte is limited, such that it can transform a maximum of three sperm nuclei into metaphase chromosomes. Furthermore, the presence of more than six sperm causes a loss of the ability of the oocyte to maintain the maternal chromosomes in a metaphase state.

Advances in the study of zygote activation in higher plants

Zygote ◽  
2020 ◽  
pp. 1-8
Author(s):  
Dong Xiao Li ◽  
Shi Jun Chen ◽  
Hui Qiao Tian
Keyword(s):  
Gene Expression ◽  
Higher Plants ◽  
Expression Patterns ◽  
Apical Cell ◽  
Sperm Nucleus ◽  
Initiation Factors ◽  
Sperm Nuclei ◽  
Quantitative Changes ◽  
And Function ◽  
Genome Activation

Summary In higher plants, fertilization induces many structural and physiological changes in the fertilized egg that reflect the transition from the haploid female gamete to the diploid zygote – the first cell of the sporophyte. After fusion of the egg nucleus with the sperm nucleus, many molecular changes occur in the zygote during the process of zygote activation during embryogenesis. The zygote originates from the egg, from which some pre-stored translation initiation factors transfer into the zygote and function during zygote activation. This indicates that the control of zygote activation is pre-set in the egg. After the egg and sperm nuclei fuse, gene expression is activated in the zygote, and paternal and maternal gene expression patterns are displayed. This highlights the diversity of zygotic genome activation in higher plants. In addition to new gene expression in the zygote, some genes show quantitative changes in expression. The asymmetrical division of the zygote produces an apical cell and a basal cell that have different destinies during plant reconstruction; these destinies are determined in the zygote. This review describes significant advances in research on the mechanisms controlling zygote activation in higher plants.

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