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

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wcre obtu~ncd from E-Monk. Germany. All other
chcmicnls were of analytical grade and obtained
from local commercial sources.
Alblno male rats of Wistar strain (45 davs old)
werc used an thc present study and oblalncd from
the Central Anhmal House. laaaharlal lnst~lutc of
Portgradu~le Mcdteal Wucatlon and Re~earch.
Pond~cherry. India. The animals wcre maintained
undcr a wcll-regulated linht and dark schedule
(12.12 hl and tcmperatureil f 3 'C and were fed
wllh sltlndard comn>erclal pcllcted fced and water
ad I~b>lum Mcthoaycblor was dissolved in acelonc
and olwe oil ([:I91 and administered orally
at the doses of 1. 10 and 100 mg kg-' body
wcipl~t wr Jay Tor 45-days. Corrsaponding groups
of annrnals wcrc admintstercd with vehtcle alone
dnd scrved us conlrol.
The animals wcrc fasted overnight, weighed and
k~llcd by urxng ancslhclic cthcr on the day following
the I;tst dosing Tcst~s, cptdidymts. scmtnal
vcs~cles and ventral prostatc were removed and
clearcd from thc adhertng tissues. The weights of
thc ttssucs wcrc rccordcd in mg as well as mg
kg - ' body wctght. Both thc testa of each animal
wcrc u\cd Tor subccllvlar irilcl~onat~on and biochcmlcal
studtcs.
M~tochoodr~al and mtcrosome-rlch fractions of
the tertls wcrc obtalncd by diffcrcntlal centrtfugatlon
mcthod as dcscrabcd prcv~ously (Lutchoumyrn~nd;knc
cl ul.. 2002a). Briefly, a 20% (w/v)
tcst~cular homogcnate was prepared in ~cecald
0.25 M sucrose solulnon with the help of a motordrtvcn
xlifss tcflon homo~enircr, The homoaenale
wa- . re8;tnfuscd -~ at 1000 ; e for 10 rnin at 4-oc to
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obtain the nuclear pellet. Mitochondrial pellet
was obtatncd by ccntr~rugtng the post-nuclear supernatant
at 1OW x I: for 10 min at 4 -C. The
m~croromc-rlch fractions were prepared by calclum
chloridc (CaClJ sedimentation method of
Kamath and Narayan (1972). Briefly, the post-ms.
tachandr~al supernatant was diluted wzth ice-cold
CaCI, (I M) so that the final concentration of
CaCI, was 0 8 molar. It was incubated at 4 'C for
10 min with occasional stirring. The sample was
then centr~iuged at IOWO x g for 10 min at 4 "C
and the m~crosome-rich fractions were obtained.
All Ihe lraclions werc washed three t!mes w~th
~ce-cold 1.15% potassium chloride solution and
d~ssolved in 0.25 M sucrose solution (1 mg protein
per 0.1 ml). The mitochondr~al and mlcrosomerich
iractions were used for biochemical studies.
Superoxlde dlsmutase (EC 1.15 1 1) was assayed
by the method of Marklund and Marklund
(1974) Br~efly, the assay mlxture contained 2.4 mi
of 50 mM tris-HCI buffer conlaming I mM
EDTA (pH 7.6). 300 PI of 0.2 mM pyrogallol and
300 PI cnzymc source. The incrcase I" absorbance
was rnoasurcd immediatelv at 420 nm arainst
blank contamin8 all the components except the
enzyme and pyrogallol at 10 s intetvals for 3 mln
on a Systronic~ Speztrophatometer. The enzyme
activtty was cxpresscd in nmolc pyrogallol oridtzcd
per min mg-' prolcin.
Catalase (EC, 1.1 1.1.6) was assayed by the
mcthod of Claiborne el al. (1985). Br~efly, the
ansay rnlxture contained 240 ml of phosphate
buffer (50 mM. pll 7.0). 10 $4 of 19 mM hydrogcn
peroxide (H20,) and 50 gl enzyme source.
The decrease in absorbance was measured Immediatcly
at 240 nm against blank conlainlng all the
components except the enzyme at 10 s intervals
for 3 min on a Systronics Spcctrophotometcr. The
enzyme actwily was expressed in lrmole oi H A
consumed per mln mg-' protein.
2.7. Glurorhione reducrose
The activity of glutathione reductase (EC.
1.6.4.2) was assayed by the method or Carlberg
and Manncrv~k (1975). BneRy, the assay mixture
contatned 1 75 rnl of phosphate buffer (100 mM.