Cannabis sativa is a cheap hallucinating agent used in different parts of the world from time unknown as a part of various religious as well as social practices. Cannabis which is a special type of Marijuana can provide temporary relief from analgesia, body pain, and in some other clinical conditions. But impacts of Cannabis on reproductive health of males and females are multi-faceted and differentially fatal. In males, Cannabis can cause changes in testicular morphology, sperm parameters (in terms of semen quality, sperm morphology, sperm mortality, and sperm motility), male reproductive hormones and finally causing reduced libido. In females, Cannabis can reduce female fertility by disrupting hypothalamic release of gonadotropin releasing hormone (GnRH), leading to reduced estrogen and progesterone production and anovulatory menstrual cycles. Current research suggest that Cannabis may negatively impact on male and female fertility conditions. However, male sterility considering the Cannabis impact is totally lacking in human as well as in sub-human primates. However, very limited studies are available on Cannabis effect on primate female reproduction considering Rhesus monkeys. Hence, further studies are needed to validate that robust findings in animal models will carry over into human experience.
- female mice
The main causative agent of marijuana/cannabinoids is the endocannabinoid. This is a neutral lipid and highly conserved molecule throughout evolutionary history . They are having different derivatives like anandamide , 2-arachidonoylglycerol  and ∆9-tetrahydrocannabinol (THC) . However, among all of the fatty acid derivatives of cannabinods or endocannabinoids (eCBs) the ∆9-tetrahydrocannabinol (THC) has now been established as the most important hallucinogenic agent of this molecule . There are literatures suggesting the role of this ∆9-tetrahydrocannabinol (THC) in regulation of functions of central nervous system and thus regulating the reproductive functions by affecting/ modulating hypothalamo-pituitary-gonadal axis (HPG-axis) 
But, all the above mentioned reported phenomenon are occurring in the central nervous system and no definitive proof has been reported till date how the enocannabinoids are affecting peripheral reproductive performances in females (in terms of gonadal activity, steroidogenesis, receptor expressions, free radical generations). Thus, aim of the present study was to note the cannabinoid (particularly endocannabinoid) induced oxidative stress and reproductive impairments in female mice specifically taking peripheral reproductive organs (ovary) in consideration.
2. Subjects and methods
2.1 Animals and maintenance
In bred adult (12–15 weeks of age), female Parkes strain mice were used for this study. Mice were maintained under hygienic conditions in a well-ventilated room with 12-h photoperiod (8 AM to 8 PM, light) with 50 ± 20% relative humidity, 25 ± 2°C temperature and were fed pelleted food (Mona Laboratory Animal Feeds, Varanasi, India); drinking water was available
Preparations of different doses of
Leaves and flowers of fresh
2.2 Purity assessment of
The dry-weight ratio of D9-tetrahydrocannabinol (THC) to cannabidiol (CBD) and the percent CBD and THC in the cannabis variant found in this region of the world has been previously reported . The proportion of high THC/CBD chemotype plants in most accessions assigned to
2.3 Experimental design
Mice were randomly allocated into three groups (groups 1–3). Each group comprised of five female mice (n = 5/group). Group 1 was treated with distilled water (vehicle treated; controls); group 2 was gavaged with 6 mg/100 g body weight/day aqueous
2.4 Collection of desired tissues
Mice were weighed before the start of experiment as well as before killing. The animals were etherized to death and blood was collected from heart. Subsequently serum was separated and was stored at −20°C until biochemical estimations of total serum cholesterol and estradiol by ELISA. Both the ovaries and uterine horns were excised, blotted free of blood and fat tissues and were weighed. The ovary on one side of the animal was fixed in Bouin’s fluid for histology and immunohistochemical localization of CB1 receptor. The contra-lateral ovary of each mouse was stored at −20°C until used for enzyme assays (for steroidogenesis, Caspase-3 and free radical parameters) and western blot analysis of CB1 receptor.
2.5 Antibodies and reagents
All of the chemicals used for the present study were of analytical grade and were purchased either from Sigma Aldrich (St. Louis, MO, USA) or from Merck (Germany). For western blot analysis, polyclonal primary antibody against CB1 receptor was purchased from Affinity BioReagents (Rockford, IL, USA, Cat No. RQ4287) and horseradish peroxidase (HRP)-linked secondary antibody was purchased from Bangalore Genei Pvt. Ltd. (Bangalore, India). For immunohistochemistry (IHC), ABC Kit was purchased from ABC staining kit (Universal Elite, Vector Laboratories, Burlingame, CA). For 3β HSD and 17β HSD assays, pregnenelone was purchased from Sigma Aldrich (St. Louis, MO, USA).
3. Experimental approaches
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 . 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 . 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 126.96.36.199) enzyme was assayed according to the protocol of Shivanandappa and Venkatesh  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.
3.4 17β hydroxy steroid dehydorgenase enzyme activity
17β HSD (EC 188.8.131.52) activity was measured by following the protocol of Jarabek et al. . In brief, 10% homogenate of the ovarian tissues were prepared in normal Phosphate Buffered Saline (PBS; pH 7.4) and 250 μl of the supernatant was mixed with 250 μl of 440 μM sodium pyrophosphate buffer (pH 10.2), 10 μl ethanol containing 0.3 μM estradiol (Sigma, St. Louis, USA) and 240 μl of 25 mg% BSA. Enzyme activity was measured after addition of 50 μl of 0.5 μM NAD to the mixture in a spectrophotometer at 340 nm against a blank (without NAD). One unit of enzyme activity was the amount causing a change in absorbance of 0.001/min at 340 nm.
3.5 Evaluation of SOD activity in ovary
Superoxide dismutase (SOD; EC 184.108.40.206) activity was assayed following the method of Das et al. . Just after sacrifice, 10% homogenates of all ovarian tissues from group 1 and set-III mice were prepared in 150 mM phosphate buffered saline (PBS, pH 7.4) and centrifuged for 30 min at 12,000 g at 4°C. The supernatant was again centrifuged for 60 min at 12,000 g at 4°C and then processed for enzymatic activity based on a modified spectrophotometric method using nitrite formation by superoxide radicals. A 0.5 ml of homogenate was added to 1.4 ml of reaction mixture comprised of 50 mM phosphate buffer (pH 7.4), 20 mM L-methionine, 1% (v/v) Triton X- 100, 10 mM hydroxylamine hydrochloride, 50 mM ethylene diamine tetraacetic acid (EDTA) followed by a brief pre-incubation at 37°C for 5 min. Next, 0.8 ml of riboflavin was added to all samples along with a control containing buffer instead of sample and then exposed to two 20 W fluorescent lamps fitted parallel to each other in an aluminum foil coated wooden box. After 10 min of exposure, 1 ml of Greiss reagent was added and absorbance of the color formed was measured at 543 nm. One unit of enzyme activity is defined as the amount of SOD inhibiting 50% of nitrite formation under assay conditions.
3.6 Estimation of catalase activity in ovary
Catalase (CAT; EC 220.127.116.11) activity was measured following the procedure of Sinha . This method is based on the fact that dichromate in acetic acid is reduced to chromic acetate when heated in the presence of H2O2 with the formation of perchromic acid as an unstable intermediate. The chromic acetate thus produced is measured calorimetrically. The catalase preparation is allowed to split H2O2 for different periods of time. The reaction is stopped at a particular time by the addition of dichromate/acetic acid mixture and the remaining H2O2 is determined by measuring chromic acetate calorimetrically after heating the reaction mixture. There is production of green color at the end of the process. Immediately after sacrifice, 20% homogenate of ovarian tissues from groups 1 to 3 were prepared in PBS (10 mM; pH = 7.0) and then centrifuged at 12,000 g for 20 min at 4°C. Supernatant was taken for enzyme estimation. About 5 ml of PBS was added to 4 ml of H2O2 (200 mM) and then 1 ml of enzyme extract was added. After 1 min 1 ml of this solution was taken in a tube and 2 ml of K2Cr2O7 (5%) solution was added. Then, it was boiled for 10 min and absorbance was measured at 570 nm. The activity of CAT was expressed as amount of H2O2 degraded per minute.
3.7 Estimation of lipid peroxidation (LPO) assay by thiobarbituric acid reactive substances (TBARS) level estimation in ovary
After sacrifice of the mice of all the groups, the ovarian tissues were dissected out on a sterile watch glass placed in ice box, cleaned from adherent tissues and processed immediately for estimation of lipid peroxidation. Ovarian tissues of groups 1–3 experimental mice were weighed and homogenized in a tenfold excess of 20 mM Tris-HCl buffer (pH 7.4) and the 10% homogenates were centrifuged for 15 min at 3000 × g at 4°C. The supernatant was subjected to thiobarbituric acid (TBA) assay by mixing with 8.1% sodium dodecyl sulfate (SDS), 20% acetic acid, 0.8% TBA and then digested it for 1 h at 95°C. The reaction mixture was immediately cooled in running water, vigorously shaken with 2.5 ml of n-butanol and pyridine reagent (15:1) and centrifuged for 10 min at 1500 × g (Ohkawa et al. ) . The absorbance of the upper phase was measured at 534 nm. Total thiobarbituric acid reactive substances (TBARS) were expressed as malondialdehyde (MDA; nmol/g tissue weight) taking 1,1,1,1-tetraethoxy propane (TEP) as standard. The standard curve was calibrated using 10 nM TEP.
3.8 Glutathione peroxidase (GPx) estimation in ovary
Glutathione peroxidase (GPx; EC 18.104.22.168) activity was assayed as described by Mantha et al. . The reaction mixture (1 ml) contained 50 μl sample, 398 μl of 50 mM phosphate buffer (pH 7.0), 2 μl of 1 mM EDTA, 10 μl of 1 mM sodium azide, 500 μl of 0.5 mM NADPH, 40 μl of 0.2 mM GSH, and 1 U glutathione reductase. The reaction mixture was allowed to equilibrate for 1 min at room temperature. After this, the reaction was initiated by addition of 100 mMH2O2. The absorbance measured kinetically at 340 nm for 3 min. The GPx activity was expressed as nmol of oxidized NADPH oxidized to NADP+ per min per mg of protein using an extinction coefficient (6.22 mM−1 cm−1) for NADPH.
3.9 Caspase 3 activity assay
Thecal cell suspension was prepared following the protocol of Sharma et al., . 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 × 6 106 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 OD405/106 cells per ml .
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 . 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 4o.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 .
3.12 Statistical analyses
The data were analyzed on Microsoft Office Excel worksheet followed by one way ANOVA. All data are expressed as mean ± SEM. The data were considered significant if
4.1 Histomorphology of ovary
The ovarian sections of both 6 mg/100 g of body weight and 12 mg/100 g of body weight showed degeneration of ovarian micro-architecture in comparison to control. There was absence of corpora-lutea in the ovaries of
4.2 Immunohistochemistry of CB1 receptor in ovary
CB1 receptors protein was demonstrated immunohistochemically in the ovaries of the control and
4.3 Body weight
We noted a significant (
4.4 Ovarian weight
We recorded the ovarian weight upon
4.5 Uterine weight
We recorded the same result in uterine weight also where
4.6 Total serum cholesterol
Serum cholesterol also showed significant dose dependent decrease (
4.7 3β HSD enzyme activity in ovary
Significant decrease in 3β HSD enzyme activity (
4.8 17β HSD enzyme activity in ovary
Significant decrease (
4.9 SOD activity in ovary
Significant increase in SOD activity was noted in
4.10 Catalase activity in ovary
Significant increase in catalase activity was noted in
4.11 Malondialdehyde level in ovary
Significant decrease in ovarian malondialdehyde levels were noted in a dose dependent manner following
4.12 GPx level in ovary
Glutathione peroxide (GPx) level was found to be significantly high (
4.13 Caspase 3 activities in ovarian thecal cells
Caspase 3 activity was assayed in the ovarian thecal cells upon cannabis treatment. We noted a significant increase of caspase 3 in the thecal cells in dose dependent manner being highest in 12 mg/100 g of body weight dose (
4.14 Serum level of estradiol
Serum level of estradiol was found to be significantly low (
4.15 Western Blot analysis of CB1 receptor in ovaries of mice
We noted a significant increase (
The present study was confined on the role of chronic
Our study, in relation to the dose dependent effect of
We noted a significant decrease in body weight, ovarian and uterine weight upon
Further, significant decrease in total serum cholesterol levels, estradiol levels in circulation, 3β HSD and 17β HSD enzyme activities in ovarian tissues upon
Thus, we may suggest that
This study, for the first time showed the effect of administration of
Financial support to SG by Council of Scientific and Industrial Research (CSIR), New Delhi, India is gratefully acknowledged.