**3.1 Histological preparations**

Ovaries were embedded in paraffin wax and serially sectioned of 6 μm using a microtome (Leica, Germany). One set of slide was prepared and was further processed for hematoxylin and eosin staining following the protocol published elsewhere [22]. The permanent slides were prepared by mounting with DPX (Distyrene Plasticizer Xylene, SRL, India), after 24 h were observed under microscope (Leitz MPV3 with photo-automat software) and were documented for general histology.

#### **3.2 Immunohistochemistry of CB1 receptor**

Immunohistochemistry for CB1 receptor was performed following the protocol published elsewhere [21]. Ovaries of both treated and untreated adult mice were

paraffin embedded, and 6 mm sections were analyzed by immunohistochemistry, for CB1receptor to show where, CB1, receptor is localized in mice ovaries and to have a generalized idea about the receptor expression pattern. For the secondary antibody and enzyme conjugates, ABC staining was used. Briefly after deparaffinization and hydration, and blocking of endogenous peroxidase with 3% H2O2 in methanol, sections were incubated with blocking serum for 1 h, followed by incubation with primary antibody (CB1 at a dilution of 1:50) for 1 h at room temperature. The sections were then washed and incubated with the biotinylated secondary antibody for 30 min at room temperature, followed by another 30 min with horse radish avidin-peroxidase conjugated. After washing, sections were incubated with the chromagen substrate (0.1% 3,3- diaminobenzidine tetrahydrochloride, DAB, Sigma-Aldrich, USA) in 0.05 M Tris buffer, pH 7.6, and 0.01% H2O2 for 10 min and then counterstained with Elrich's hematoxylin. The permanent slides were prepared by mounting with DPX (Distyrene Plasticizer Xylene, SRL, India), after 24 h were observed under microscope (Leitz MPV3 with photo-automat software) and were documented.

#### **3.3 Estimation of total serum cholesterol**

The total serum cholesterol was estimated by commercial cholesterol estimation kit following manufacturer's protocol (Span Diagnostics, Surat, Gujarat, India).

3β hydroxy steroid dehydrogenase enzyme activity:

3β HSD (EC 1.1.1.145) enzyme was assayed according to the protocol of Shivanandappa and Venkatesh [23] using ovarian homogenate. Ten percent tissue homogenate was prepared in 0.1 M Tris-Cl buffer (pH 7.8). The homogenate was centrifuged at 12,000 g at 4°C and the supernatant was used as the source of enzyme. The enzyme was assayed in 0.1 M Tris-Cl buffer (pH 7.8) containing 500 mM NAD, 100 mM pregnenolone as substrate and enzyme (50 ml) in a total volume of 3.0 ml and incubated at 37° C for 1 h. The reaction was stopped by the addition of 2.0 ml of phthalate buffer (pH 3.0) and the absorbance was noted at 490 nm. The enzyme activity was calculated from the standard curve of NADH and expressed as nmol NADH formed/h/mg protein.
