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

The plates were dried by inverting on absorbent material. Streptavidin-Enzyme
Conjugate Solution (200 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. The plates were dried by inverting on the absorbent material.
TMB chromogen solution (100 pL) was added to each well. The wells were
incubated while shaking at a fast speed on an orbital microplate shaker, for 10 min at
room temperature. Stopping solution, 0.2 M sulfuric acid, (100 pL) was added to
each well and absorbance of the solution in the wells was read within 30 min using a
microplate reader set at 450 mn. The mean absorbance for each standard, control and
experimental senun samples were calculated. Using a linear-linear graph paper mean
absorbance readings were plotted for each of the standards along the y-axis versus the
estradiol concentrations in pg/ mL along the x-axis. The best-fitting curvc through the
mean of the duplicate points was drawn. The estradiol concentrations of the controls
and cxpcrimcntal serum samples wcrc determined Ikon1 tho standard cilrvc hy
matching their mean absorbance readings with the corresponding estradiol
concentrations.
3.14 Activities of steroidogenic enzymes
The activities of 3P-hydroxysteroid dehydrogenase and I7P-hydroxysteroid
dehydrogenase were assayed in testis after necessary standardization experiments. A
5% testicular homogenate was prepared in cold normal saline and centrifuged at 800 g
for 20 min at 4°C. The supernatant was used for assaying steroidogenic enzymes in
testis.
3.14.1 Assay of 3p-bydroxysteroid dehydrogenase
The activity of 3p-hydroxysteroid dehydrogenase (EC 1.1.1.5 1) was
determined by the method as described by Bergmeyer (I 974). The reaction mixture
contained 600 pL of 100 pM pyrophosphate buffer @H 9.0). 200 pL of 0.5 ph4