**3.9 Caspase 3 activity assay**

Thecal cell suspension was prepared following the protocol of Sharma et al., [29]. In brief, thecal cell suspensions from all the groups were prepared by mincing the entire ovary in ice-cold 1 PBS, at 4°C. After washing, cell pellets were collected by centrifugation at 500 g for 10 min at 4°C and the supernatant was gently removed. Cell pellets were lysed by the addition of 50 ml of cold lysis buffer (5 mM Tris, 20 mM EDTA, 0.5% Triton-X 100, pH 6.0) per 2 6 10<sup>6</sup> cells and incubated on ice for 10 min. Lysates were centrifuged at 10, 000 g for 1 min at 4°C, and the supernatant was transferred to a fresh tube and processed for caspase-3 (EC 3.4.22.xx) activity using a caspase-3 colorimetric assay kit, according to manufacturer's instructions (R&D Systems, Inc. MN). Each enzymatic reaction, carried out in a 96-well flat bottom microplate, required 50 ml cell lysate, 50 ml reaction buffer, and 5 ml caspase-3 colorimetric substrate (DEVD-pNA). The plate was incubated at 37°C for 2 h with a substrate blank and sample blank. At the end of the incubation period, the absorbance of enzymatically released chromophore p-nitroanilide (pNA) was read at 405 nm in a microplate reader (Tecan, Spectra II-micro-ELISA plate reader, Austria). Caspase-3 activity was determined by comparing the absorbance or optical density (OD) of pNA from apoptotic samples with the untreated control and expressed as fold increase in OD405/10<sup>6</sup> cells per ml [29].

#### **3.10 Serum level of estradiol**

Estradiol was assayed using ELISA kit (Biotron Diagnostics Inc., USA) according to manufacturer's protocol. The coefficient of intra- and inter-assay variation was less than 4.1 and 6.4%, respectively. The analytical sensitivity was 10 pg/ml.

#### **3.11 Western blot analysis of Cannabinoid receptor 1 (CB1) analysis**

The ovarian tissue protein pooled from six mice was extracted as described earlier [30]. For western blot analysis, 10% ovarian homogenate was prepared. Equal amounts of proteins (50 mg) determined by Bradford's method were loaded on SDS PAGE (10%) for electrophoresis. Thereafter, proteins were transferred electrophoretically to nitrocellulose membrane (NC; Sigma-Aldrich, USA) overnight at 4<sup>o</sup> .C NC was then blocked for 60 min with Tris-buffered saline (TBS; Tris 50 mM, pH 7.6) and then incubated with primary antiserum (CB1 at a dilution of 1:250) for 1 h. Then, membranes were washed for 10 min each (three washes) in TBS-Tween 20. Then, NC membrane was incubated with secondary conjugated with serum immunoglobulin (1:500) for 30 min and then washed in TBS for 10 min (three times). Signals were detected using an ECL kit (Bio-Rad, Hercules, CA). Blot for each protein was repeated for three times. The densitometry analysis of blots was performed by scanning and quantifying the bands for density value by using computer-assisted image analysis (Image J 1,38X, NIH). The densitometry data were presented as the mean of the integrated density value SEM. A pre-stained

multicolor broad range marker (Spectra TM multicolor broad range marker; 10 to 260 kDa x SM-1841; Fermentas, MD, USA) was also run along with sample proteins to clarify the position of band obtained as published elsewhere previously to detect the specificity of the bands [30].
