**5. Discussions**

The present study was confined on the role of chronic *Cannabis* induced oxidative stress and reproductive impairment in female mice. In the recent years, there are several literatures available depicting the role of *Cannabis* in neurodegeneration [32], neuro-myopathy [33] and different other neurological disorders [34]. But, till date there are no data or reports are available depicting the role of *Cannabis* treatment in regulating/modulating the female reproduction, however, it had been predicted from prolonged time that *Cannabis* is potent enough to interfere in reproduction in males [35] and females.

Our study, in relation to the dose dependent effect of *Cannabis* treatment in the female reproduction is the preliminary and elaborated study depicting the deleterious and detrimental effects of *Cannabis* in female reproduction. Our study is divided into two different parts addressing the role of *Cannabis* in reproductive impairments in female mice due to oxidative stress and loss in the functions of steroidogenesis.

We noted a significant decrease in body weight, ovarian and uterine weight upon *Cannabis* treatment suggesting the first clue in reproductive impairment upon *Cannabis* treatment. The results were further supported by degeneration in ovarian histomormhology and increase in expressions of CB1 receptors in ovaries of different treatment groups. Cumulatively, the histological and immunohistochemical data suggest a dose dependent impairment in ovarian as well as reproductive functions which are in agreement with previous reports in where *Cannabis* causes reproductive impairment in males [36]. We have also studied the different aspects of free radical as well as reproductive enzyme activities (3β HSD and 17β HSD). The SOD, catalase and GPx levels were significantly high in ovary tissues where as MDA level was significantly low. The increased results of free radical scavenging enzyme activities suggest the reproductive impairment in mice is may be due to high generation of free radicals and also due to different physiological malfunctions which are yet to be traced out [37–40].

Further, significant decrease in total serum cholesterol levels, estradiol levels in circulation, 3β HSD and 17β HSD enzyme activities in ovarian tissues upon *Cannabis* treatment were noted. Thus, we may suggest that upon *Cannabis* treatment reproduction in females was impaired by *Cannabis* treatment by generation of free radicals in female reproductive tissues. The results were also discussed in light of apoptosis in thecal cells by Caspase 3 activity assays and it was found to be significantly high in different doses of *Cannabis* treatments. To delineate the possible molecular mechanism of *Cannabis* function in ovary, we checked the CB1 receptor expression in ovarian tissues and we also found that the CB1 receptor expressions were significantly high in both the 6 mg/ 100 g of body weight and 12 mg/100 g of body weight groups which are in agreement with the reports published earlier [41–43].

Thus, we may suggest that *Cannabis* treatments were not only impairing the reproduction in females but also chronic duration of doses is responsible for high fecundity in terms of reproductive malfunctions.
