Effect of Various Factors on DNA of Human Sperms

found of Various Factors on desoxyribonucleic acid of adult male spermatozoanatozoanatozoonatozoon cellular teleph wholenesssIn vitro try out of the proceeding of various factors on desoxyribonucleic acid of Human spermsDave Avani1, Jainist NK1, Patel Himanshu 2, Patel Madhuri 2, Bhatt Vidisha 2, Patel Komal2, Mallick Sarada3, *Srivastava Pradeep 3AbstractAim Effect of various clinical mix and environwork forcetal conditions were studied for the in vitro fragmentation of the human sperm deoxyribonucleic acid, as well on the quality of semen takes. Method The semen samples obtained from antithetical males were then treated further to check the make of the chemicals taken into consideration. The effects were studied through sperm chromatin dispersion rill. Results Primary results showed that the antibiotic tetracycline was the most effective chemical ca utilize desoxyribonucleic acid deterioration, as comp bed to the other chosen compounds. Conclusion The study con cludes that the tetracycline drug is more efficient then the others in causing fragmentation of the DNA.Keywords Sperm DNA, Fragmentation, tetracycline, Sperm Chromatin Dispersion block out.INTRODUCTIONThe seed line is the most natural of all and can be considered as a good view for the measurement of the effects ca employ by the compounds of choice 1-4. Today sterility is conjugated to m any(prenominal) reasons such as mutation in the sperms or the egg, irradiation, and accepted chemicals such as food elongates, packaging materials, heat etc. umteen articles gift been put forward proving the adverse effects of these chemicals on the germ line especially the sperms and its DNA. The paternal genome in mammalian spermatozoa is condensed in a manner that is specific to the cell type presumably to protect the DNA during the transit from the male to the oocyte prior to stuffing. Damaged DNA has been observed in testicular, epididymal and ejaculated sperm. Temporary nicks have been observed in the sperm which were, linked to the topoisomerases occupation, avail histone-protamine replacement, further if these nicks are not fixed they would evolve into DNA fragmentation on mature sperm 5.Bisphenol A (BPA) used to make plastics and paste resins mainly comes through diet is an radical compound with the chemical linguistic rule (CH3)2C(C6H4OH)26-8. It is part of the bisphenols group of chemical compounds with two hydroxyphenyl chromosome mappingalities. It is a twineless consentient that is soluble in organic solvents, but poorly soluble in water. Many studies also have shown that BPA has estrogenic activity in several(prenominal) in vitro and in vivo preparations 8-10.Monosodium glutamate, also known as sodium glutamate or monosodium glutamate, is the sodium salt of glutamic acid, one of the most abundant naturally occurring non-essential amino acids11-13. Industrial food manufacturers market and use MSG as a flavour enhancer because it balances, blends and rounds the total perception of other tastes. Many experiments have been carried out which have shown the harmful effects of excessive intake of MSG on the development of seminiferous tubules as well as the spermatids14-15.The tetracyclines are a family of antibiotics that inhibit protein synthesis by preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor (A) site. Tetracyclines are broad-spectrum agents, let oning activity against a wide range of gram-positive and gram-negative bacteria, a common organisms commonly apt(p) in treatment10, 16.Many studies in the past indicated that when rats were administered a epoch-making amount of Tetracycline on daily basis the physicals showed a change magnitude level of testosterone ultimately affecting the development of the leydig cell. No direct kind has been observed till date on changes in the humans but many studies are being carried out to study the detrimental effects of tetracycline on human sperm cells and its DNA17 ( name.1).Fig.1.Molecular mental synthesis of compounds a) Molecular structure of Bisphenol A, b) Molecular structure of Monosodium Glutamate c) Molecular structure of Tetracycline.MATERIALS AND METHODSMaterialsSample analysis was make for the sanitary men between the ages 25-30 yrs. The inclusion criteria were only vigorous men with the age between 25-30 yrs considered. On the hand exclusion criteria of the male were they should impeccant from various diseases like Diabetes, Blood Pressure, Tuberculosis, Sexual Dysfunction Cardiac problems.The samples cool from Stem Cure Pvt Ltd, Centre for Reproductive Medicine Stem cell Development, Ahmedabad, India. The study approved by the Ethical Committee of the Department of biography Sciences, School of Sciences, Gujarat University, Ahmedabad, Gujarat (India).Sample preparation 10 semen samples of absolutely wellnessy male were collected at a collection centre in a sterile jar and were brought to the laboratory. Dir ect swim-up technique was utilised for preparing the sperm cells for the analysis18. The sperm debris including the dead sperm cells was removed prior using by centrifuging the sample in a Phosphate Buffered Saline (phosphate polisher tooth root) specialty at 1000 rpm for 10 minutes19. The sperm pellet obtained was overlaid with PBS and incubated at 37oC and the supernatant containing the motile sperms were used for the assay. Effect of heat was equivalence with normal cells discussed later(prenominal) in the discussion section.Chemicals Bisphenol A (BPA), Tetracycline, Monosodium Glutamate (MSG) , concentrated HCl, 0.4M Tris, 1% mercaptoethanol, 50mM EDTA, 1% SDS (sodium dodecyl sulphate), pH 7.5 was used for making Lysis I, 0.4M Tris, 2M NaCl, 1% SDS, pH 7.5 was used for making Lysis II20, 0.09M Tris borate, 0.002M EDTA, pH 7.5 was used for making the wash buffer and double-dyed(a) ethanol. 0.4 gm, 0.6 gm, 0.8 gm of Tetracycline, MSG, BPA were dissolved in 100 ml of Doubl e Distilled pissing (DDW) to get 4%, 6% and 8% of absorption. Individual studies of samples were conducted using idiosyncratic chemicals separately. sensible analysis of Semen Semen sample were physically analysed by and by the liquefaction, and viscosity were checked by observing the droplets falling from a liquid plastic pipette.Vitality Staining The semen sample was studied for the presence of any unwanted materials using vitality placeing test. The sample was complicated and a smear was prepared on a glass splay along with eosin-nigrosin stain and was observed under the microscope at 100X magnification by and bywards the smear dried completely. The live sperm heads were seen white in colour while the dead sperm heads were stained as pink.Analysis of DNA fragmentation using the SCD Test Slides precoated with 1% agarose were used as a base slide. Final concentration of 15-20 million sperms were mixed with 1% low melting point agarose overlaid onto base slide and covered with coverslip21. The slides were unbroken at 4C to get the gel solidified and later the coverslips were removed carefully. No reduction was observed, however minute reduction was in that location maintained with PBS.Chemicals of different concentrations i.e. 4%, 6%, and 8% (Tetracycline, MSG and BPA should be prepared freshly prior to the analysis). The slides were kept in these individual solution for time 1.5hrs and 3hrs respectively, study effect of heat was made by modify the agar mixed sample containing the slides of sperm the agar clean was het up(p) at 45oC 55oC and 60oC.After the incubation period the slides were immediately removed and immerse in 0.08N HCl for 7minutes. The slides were then kept for Lysis in two cycles of 10 minutes followed by 5 minutes respectively. The slides were then washed with PBS buffer and dehydrated with 70%, 90% and 100% ethanol for 2minutes respectively. The slides were allowed to dry and are then observed under visible light-colored us ing geimsa stain 5, 22.RESULTSemen Analysis The result of physical analysis of semen is as shown in Table 1. The semen samples studied were free of any contaminants and appeared normal. The pre and post wash count were also carried out which showed that the semen samples were perfectly healthy. The viscosity and the volume of each sample under study were indoors the normal range as suggested by the world health administration standard.Analysis of DNA fragmentation using the SCD (Sperm Chromatin Dispersion) TestThe SCD test carried out depict the effect of the chosen compounds on the sperm DNA which are acted as under.(A) Effect of hot up- Sperm sample were subjected to heat treatment in water bath for 1.5 hrs at the temperature of 45oC and 55oC, where normal temperature of 28oC was used as control. The results are shown in fig 2.0.(B) Effect of Chemicals-The studies were made on the percentage injure observed by and by chemical treatment to the sperm cells for 1.5 hrs and 3hr s of incubation time. Fig 3.0 and 4.0 depict the effect of tetracycline, BPA and MSG on sperm cells for 1.5 hrs and 3.0 hrs of incubation time respectively. Sperm cells without chemicals but buffer solution were taken as control. Various concentrations of chemicals viz, 4 8% was taken for the study.It was observed that tetracycline causes the maximum constipation of about 92.4% afterwards an incubation time3hrs at 8% concentration, while the treatment with heat, monosodium glutamate and bisphenol exhibit a maximum damage of 55.6% at 55C, 55% and 44% respectively after an incubation time of 3hrs at 8% concentration.Table.1. Physical analysis of semenFig.2.Effect of Heat Treatment on sperm cellsFig.3.Effect of Chemicals on sperm cells (incubation time 1.5h)Fig.4.Effect of Chemical on sperm cells (incubation time 3 h)Table.2.Anova MethodDISCUSSIONStatistical Data Analysis The table 2.0 depicts below is the statistical representation of the data obtained after the SCD test. Anova tes t was applied using t-Test Software (Excel). It shows that the data obtained from the SCD are material and valid. P=0.05, It was large at 55C. Control groups are healthy individuals where sperm cells were preserved and treated at 28C without additive chemicals (MSG, BPA, and Tetracycline). Samples collected from the normal subjects. The table exhibit that F value of Tetracycline Bisphenol A and MSG are 22.24, 13.435 and 14.405 respectively the observation are significant Thus it can be said that the hypothesis given by the author is correct i.e. It was significant. Tetracycline gives a extravagantlyer percentage of damage to the DNA of the sperm cells in vitro.Image Analysis of Damage after the TreatmentThe figures.5 (a to f) given were obtained after under a visible light observation. The figures below show the extent of damage caused by the treatment given to the cell after 3hrs of incubation.The halos seen represent the extent of damage i.e. large halos represent less damage w hile the cells without any halos represent the most shamed cell.The control slide cells have a normal halo most which depicts healthy DNA while the cells seen in tetracycline treated cells lose the halo which shows that the DNA of the sperm cells have been modify to a greater extent. The slide treated with Bisphenol A shows a less amount of damaged cell in comparison to that of Tetracycline. The least damage has been in the sample treated at 45C shown by large halos close to the cells. Hence from these figures it can be said that the tetracycline treatment produces the highest damage to the DNA of sperm cells.Fig.5.Image Analysis of Damage a) Control Slide, b) Tetracycline at 8% concentration after 3hrs of incubation, c) Bisphenol A at 8% concentration after 3hrs of incubation, d) Monosodium Glutamate at 8% concentration after 3hrs of incubation , e) Effect of temperature at 45oC f) Effect of temperature at 55CCONCLUSIONThe study concludes that the drug tetracycline used for a ntibiotics for patients is proven to cause damage to the sperm DNA along with monosodium glutamate which is a flavour enhancing substance, as well as Bisphenol A which is a component of food packaging materials such as plastic bottles, feeder bottles etc. Though the exact mechanism by which the DNA is being affected is not known it can be said that exposing the sperm with the highest concentration of the above considered chemicals can be one of the many reasons which cause DNA damage which may lead to infertility in the present lifestyle. 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