ON TESTIS AND EPlDlDYMlS OF RATS - Pondicherry University ...

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Approximately 10 FL of thoroughly mixed diluted specimen transferred to each of the counting chambers of the hemocytometer and allowed to stand for 5 min in a humid chamber to prevent drying out. Sperm cells sedimented during this time and were then counted with the help of light microscope at 200 x. Complete spermatowa, head with tail. were counted. 3.1 2 Histometric studies The testicular tissue was fixed in Bouin's fixative immediately after isolation. After dehydration in alcoholic series and cleaning in xylol, the tissue were embedded in paraffin wax. Sectiow were made with 5 pn thickness. The sections were stained with hematoxylin-eosin and examined under a light microscope. 3.12.1 Tubular nod lumen diameter measurements The diameter of the seminiferous tubule and lumen were measured with eyepiece graticules that have been calibrated with a stage micrometer. Only tubules that appeared round were considered for tubular and lumen diameter measurements. 3.13 Quantitative determination of serum hormone levels Serum levels of follicle stimulating hormone (FSH), luteiniziny hormone (LH), prolactin. testosterone and esrradiol were determined by enzyme linked imrnunosorbant assay (ELISA) using kits from Diagnostic Systems Laboratories, Inc. Webstcr, Texas, USA. The assays were done strictly according to the procedure given along with the kits 3.13.1 Quantitative determination of serum FSH Serum levels of FSH were determined strictly according to the procedure given along with kit. In principle FSH ELlSA kit is an enzymatically amplified 'twostep' sandwich-type immunoassay. In the assay, standards. controls and experimental serum samples are incubated in microtitration wells, which have been coated with
anti-FSH antibody. After incubation and washing, the wells are treated with another anti-FSH detection antibody labeled with the enzyme horseradish peroxidase (HRP). After a second incubation and washing step, the wells are incubated with the substrate tetramcthylbenzidine (TMB). An acidic stopping solution is then added and the degree of enzymatic turnover of the substrate is determined by absorbance measurement at 450 nm. The absorbance measured is directly proportional to the concentration of FSH present in the serum samples. A set of FSH standards is used to plot a standard curve of absorbance versus FSH concentration from which FSH concentrations in the experimental serum samples can be calculated. Before starting the assay all the reagents and serum samples were brought to room temperature and were thoroughly mixed. The assay consisted of standards containing concentrations of approximately 0, 1.5, 4.5, 15.45 and 150 mIU/ mL FSH (see Appendix 1.3), and controls containing low (Level 1) and high (Level 11) concentrations of FSH and experimental serum samples. All the assays were performed in duplicate and internal and external quality controls were maintained. The microtitration strips were marked and 100 pL of the standard. controls and experimental serum samples were added to the appropriate wells and incubated while shaking at a fast speed (500 to 700 rpm) on an orbital microplate shaker (P- Multi~ope ELISA Reader) for 60 min at room temperature. Antibody-Enzyme Conjugate solution (100 pL) was added to each well and incubated while shaking at a fast speed on an orbital microplate shaker for 30 min at room temperature. The wells were aspirated and washed five times with the wash solution using an automatic microplate washer (Labsystems Well Wash 4). The microtitration plates were dried by invening on absorbent material. TMB chromogen solution (100 pL) was added to each well and incubated while shaking at a fast speed (500 - 700 rpm) on an orbital microplate shaker for 10 min at room temperature. Stopping solution, 0.2 M sulfuric acid, (100 a) was added to each well and absorbance of the solution in the wells was read within 30 min using a microplate reader (P-Multiscope ELISA Reader) set at 450 nm. Mean absorbances for standard, control and experimental serum samples were
Page 1 and 2: FUNCTIONAL STUDIES ON THE EFFECTS O
Page 3 and 4: ACKNOWLEDGEMENTS I wish to express
Page 5 and 6: TABLE OF CONTENTS Page No ACKNOWLED
Page 7 and 8: TABLE OF CONTENTS (Continued) Deter
Page 9 and 10: TABLE OF CONTENTS (Continued) Effec
Page 11 and 12: TAULIC OF CON'I'ISN'I'S (C'untinued
Page 13 and 14: xi LIST OF TABLES (Continued) I'agc
Page 15 and 16: Table 24a Table 24b Table 24c Table
Page 17 and 18: Fig. 1 Fig. 2 Effect of TCDD on the
Page 19 and 20: Fig. 12c Fig. 12d Fig. 13 Fig. 14a
Page 21 and 22: LIST OF FIGURES (Continued) Fig. 23
Page 23 and 24: LH M PL mg mRNA N AD NADH NADP NADP
Page 25 and 26: layers, an outer layer of visceral
Page 27 and 28: tubular epithelium and continually
Page 29 and 30: lmn~unization of monkeys against FS
Page 31 and 32: and this reaction is catalyzed by t
Page 33 and 34: extratubular environment or synthes
Page 35 and 36: the most active in protein secretio
Page 37 and 38: 1958). A rapid fall in serum testos
Page 39 and 40: have also been reported in the rats
Page 41 and 42: Effect of TCDD on heart muscle has
Page 43 and 44: 1.4 Effect of oxidative stress on m
Page 45 and 46: 2 SCOPE OF THE PRESENT STUDY There
Page 47 and 48: 3 MATERIALS AND METHODS 3.1 Animals
Page 49 and 50: Subgroup III: Administration of TCD
Page 51: choppcd with the help of sharp razo
Page 55 and 56: lust sped (500 to 700 rpm) on an or
Page 57 and 58: 3.13.4 Quantitative determinntiun u
Page 59 and 60: The plates were dried by inverting
Page 61 and 62: centrifuged at 3,500 g for I5 min o
Page 63 and 64: 3.17 Preparation of tiasue humogena
Page 65 and 66: supcrnutant was taken. Hlunk was pr
Page 67 and 68: 3.21.4 Assay of glutathione peroxid
Page 69: 4 RESULTS 4.1 Administration of TCD
Page 72 and 73: seminiferous tubules was found to b
Page 74 and 75: fractions of rat testis as compared
Page 76 and 77: 4.1.11.1 Effect of TCDD on the prod
Page 78 and 79: 4.2 Co-administration of TCDD and v
Page 80 and 81: 4.2.8 Effect of co-administration o
Page 82 and 83: 4.2.11.5 Effect of co-administratio
Page 84 and 85: Table I b. Effect of TCDD on the we
Page 86 and 87: Table 4. Effect of TCDD on the seru
Page 88 and 89: Table 7b. Effect of TCDD on the pro
Page 90 and 91: Table 8c. Effect of TCDD on the lev
Page 92 and 93: Table 9d. Effect of TCDD on the act
Page 94 and 95: Table 13. Effect of TCDD on the ant
Page 96 and 97: Table 15. Effect of TCDD on the lev
Page 98 and 99: Table 16c. Effcct of TCDD on the an
Page 100 and 101: Table 18a. Effect of co-administrat
Page 103 and 104:
Table 21. Effect of co-administrati
Page 105 and 106:
Table 24b. Effect of co-administrat
Page 107 and 108:
Table 25c. Effect of co-administrat
Page 109 and 110:
Table 26d. Effect of co-administrat
Page 111 and 112:
Table 30. Effect of co-administrati
Page 113 and 114:
Table 32. Effect of caadrninistrati
Page 115 and 116:
Table 33c. Effcct of co-administrut
Page 117 and 118:
5 DISCUSSION 5.1 Significance of th
Page 119 and 120:
Measurement of weights of the acces
Page 121 and 122:
compartment of the cells (Chainy el
Page 123 and 124:
the reduction in daily sperm produc
Page 125 and 126:
eported in the TCDD-treated rats (M
Page 127 and 128:
lilnction is to ililcrccpt lipid pc
Page 129 and 130:
in the animals administered with TC
Page 131 and 132:
protective effects on serum testost
Page 133:
suygcstccl that vivamin 1: conld im
Page 136 and 137:
Fig.4~. ENect of TCDD or TCDD and v
Page 138:
tE W d In w it I I (,OCX) 3s PUB (,
Page 142 and 143:
Fig. 7c. Effect of TCDD or TCDD and
Page 145 and 146:
u 3 f ~ = m w * N O .- ~Slew aew en
Page 147 and 148:
Fig.lOb. ENmt of TCDD or TCDD and v
Page 151:
Fig. 1Ir Effect of TCDD or TCDD md
Page 158 and 159:
Fig. 14c. Effect of TCDD or TCDD an
Page 160 and 161:
Fig. 16b. Effect of TCDD or TCDD an
Page 164 and 165:
Fig.18~. EKect of TCDD or TCDD and
Page 166 and 167:
1 1.2 l4 Fig. 19b. ENect of TCDD or
Page 168 and 169:
Fig. ZOb. Elkt of TCDD or TCDD and
Page 171 and 172:
Fig. 21. Effect olTCDD or TCDD and
Page 173 and 174:
Fig. 22c. Effect of TCDD or TCDD an
Page 175 and 176:
Fig. 24b. Eflect ofTCDD or TCDD and
Page 177 and 178:
7 REFERENCES Aitken RJ, Irvine DS,
Page 179 and 180:
Chitm KC, Latchoumycandanc,C, Mathu
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Flsher JS, Turner KJ, Brown D, Shar
Page 183 and 184:
Hornsby PJ. Regulation of cytochrom
Page 185 and 186:
Latcboumycandane C, Chitra KC, Math
Page 187 and 188:
Moore RW, Panona JA, Bookstaff RC,
Page 189 and 190:
Rommerts FFG, Cooke BA, Van Der Kem
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Slaunwhite WR, Burgett MJ. h virro
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1.1 Preparation of dosing solution
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1.4.6 Wash concentrate One bottle,
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1.7.3 Estradiol controls Two vials.
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1.1 1.2 Orcinol(6%) 6 g of orcinol
Page 201 and 202:
1.16.2 Horseradish peroxidase (8 Un
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1.20 Superoxide dismutase 1.20.1 Tr
Page 205 and 206:
1.24.2 Thiobarbituric acid (TBA; 0.
Page 207 and 208:
Chronic effect: of enclosulfan on t
Page 209:
enbw1h1.W mu IUY Wcab unpiliml~t at
Page 212 and 213:
cbniuis 11~11 b"nci:~lc ~wlive oxyg
Page 214 and 215:
DNA, RNA ;uxl Wcin wcrr abarval, wh
Page 216 and 217:
Mt~~m(10-lZsu&)vacob- Wfmn Ihehml ~
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New Delhi, llrliil Ib SCI~KK Rcm& M
Page 220 and 221:
tiii~lc rcprmluction or rut (Cllitr
Page 222 and 223:
wciglit (b.wt.) per day for 45 days
Page 224 and 225:
0.1 00 01 " 0.8 I . .lo Dl* rcnn IW
Page 226 and 227:
Chilr:~. K.C.. L!olnn!myci!~dum. C.
Page 228 and 229:
C. hteboumyeradlos . KC. CMtn . P.P
Page 230 and 231:
I. 1 Ilal 01 2 1.7 # I c I ~ ~ L I
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Fin. L Corrclatwn bclwfcn "perm cou
Page 235:
Iionshii nepmdvctivc mxicity ad upt
Page 240 and 241:
o. Glutathione reductage mg pmWn mg
Page 242 and 243:
IOWAME -. Tor. #558517 PME: 9 SESS:
Page 244 and 245:
JOBNAME: Rqm. Tm. M5W7 PAOB: 11 SBS
Page 246 and 247:
t 02 ELSEVIER Toricology OM) (2W1)
Page 248 and 249:
---- - wcre obtu~ncd from E-Monk. G
Page 250:
3.2. A,III~AILNI syrlo~t of ,nIroch
Page 253 and 254:
Uc,#!dy. SC. N;tdcru. S , 1% Conlrb
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