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108 DISCUSSION Steroid use by the athletes has continued to be increase on despite the efforts of various of various sports organizations to crub a practice which had previously been restricted to weight lifters and professional body builders. Recreational body builders attending gymnasia are abusing steroids, but the frequency and pattern of use and the associated problems are less well known. In the present study, we have observed the effects of the short term (acute) and long term ( chronic ) use and withdrawl effects of the use of androgenic anabolic steroids on the morphology of testes in albino rats was observed as the rats appears to be a more suitable animal in studying the roles of androgenic anabolic steroids within the male reproductive system. This study was carried out by observing and recording the changes in behaviour, body weights of the animals and the relative weight and the gross appearance of the testes. The morphological and the histological changes in the spermatogenic cells, with reference to their number and diameter, the number of seminiferous tubules and the thickness of germinal epithelium, as well as the number and the diameter of the interstitial cells nuclei were evaluated with the help of light microscopy. Numerous studies on androgenic anabolic steroids have shown that it possesses potent toxicity on male sexual organs – the testis, by causing the disturbances of spermatogenesis in albino rats (Lee and Kim, 1985; Anthony, 2006; Shittu et al., 2006; Abel et al., 2008 ). The androgenic anabolic steroids are known to elevate oxidative stress in the testes 109 (Aydilek et al., 2004). Understanding the mechanism of action of testicular toxicity is important in elucidating not only the cause of testicular dysfunction in human exposed to the androgenic anabolic steroids but also its prevention. Another frequent adverse event relating to sexual function in males administering anabolic steroids is reversible azospermia and oligospermia ( Alen and Suominen, 1984; Shurmeyer et al., 1984 ). As exogenous androgen use increases, endogenous testosterone production is reduced. As a result, testicular size is reduced within three months of androgen administration ( Alen and Suominen, 1984 ). During this time the risk of infertility is increased. The effect of anabolic steroids on the testis results from the negative feedback of androgens on the hypothalamic-pituitary axis and possibly from local suppressive effects of excess androgens on the testis (Schumeyer et al., 1984; Pope et al., 1994; Turek et al ., 1995 ; Clark et al., 1997; Wroblewska, 1997; Nagata et al., 1999; Torres et al., 2001) Holma (1997) and Alen, (1984), postulated that androgenic anabolic steroid use can lead to hypogonadotropic hypogonadism. The resulting effects include a decline in sperm count, abnormal morphology and testicular atrophy. This study is basically directed to study the effects of short and long term use of androgenic anabolic steroids over the spermatogenesis and its relation with the inhibition of hormones which are required for male reproductive system and reversibility of these effects after withdrawing their use. 110 In the present study, injection testoviron has been used in group B at a dose of 400 mg / kg body weight for four weeks. The animals in group C were given the same dose for fourteen weeks. The group D animals were left untreated after fourteen weeks for another fourteen weeks (Feinberg et al., 1997; Clark et al., 1997 ) to evaluate whether after withdrawing the drug the changes can return back to normal or not. The observations were compared with group A animals (i.e. age and weight matched normal control) and among each other. As there is very little histological work has been done so we planned to see the different parameters of the seminiferous tubules and theinterstitium and confirmed our finding in a very sophisticated method i.e. scanning electron microscopy which is possibly one or the only research work done by any researcher. In the present study, it was observed that there was gain in weight in all the groups throughout the experimental period more in groups B and C, probably due to the anabolic effects of androgenic anabolic steroids and probably from water retention in the body and decreased high density lipoprotein among others ( Bates et al., 1987; Wilson, 1988; Wagner, 1989; Smith et al., 1992; Bhasin et al., 1996; Bhasin et al., 1997; Pope et al., 1994; Anthony, 2006 ). Regarding the relative weight of the testes, the testoviron treated group showed evidence of significant reduction in relative testicular weight in acute group and more in chronic group as compared to control. These differential changes in testicular weights observed are well correlated with the seminiferous tubular profiles of the testis for the different groups in other studies, such 111 effects has been reported by Squires et al., (1982); Noorafshan et al., (2005); Shittu,(2006); Shittu et al., (2006); Mesbah et al., (2007); Mesbah et al., (2008). Regarding the weight gain Shittu et al., (2009) that mean average weight gain in treated and control animals were not significantly different but he founded that there was significantly (P<0.05) in the proviron treated group as compared to the control group. The present study demonstrated the partial arrest of spermatogenesis as indicated by the decrease in the number and layers of spermatogonia, spermatocytes, spermatids and spermatozoa in animals in group B and more in group C when compared with the age matched control group. These changes occurred in the stage VIII of the cycle of the seminiferous epithelium. These may be attributed to the action of androgenic anabolic steroids on the dividing spermatogenic cells. As most AAS and high doses of testosterone intake exert an inhibitory effect on the hypothalamo – hypophyseal - testicular axis with a resultant suppression of in the normal testicular function which may further lead to a reduction in testosterone production, a decreased spermatogenesis, and a testicular atrophy as reported by Bra''mswig et al.,(1984); Mauro, (1988); Prader and Zachmann, (1978); Van dekerckhove et al., (2000); Dohle et al., (2003). The count of the seminiferous tubules per field was significantly increased in animals in group B and more in group C. This increase may be attributed to the loss of germ cells, resulting in the shrinkage of the tubules. Our observations closely correspond to the conclusion drawn by Squires et al., (1982), who stated that all the anabolic steroid treatment reduced the number 112 of developing germ cells, resulting in increase number of seminiferous tubules. These finding of the seminiferous tubules were also confirmed by the scanning electron microscopy (SEM) shown in figures 15 – 20. The diameter of the seminiferous tubules and the thickness of the germinal epithelium was also decreased significantly in both the treated groups when compared with that of control group. Compared to the control group, the seminiferous epithelium of the treated animals was disrupted with broad spaces between the cellular components showing the presence of copious vacuoles frequently associated with degenerating germ cells, as explained by the Mesbah et al., (2008) who observed that there was disruption of the seminiferous epithelium with broad spaces between the cellular components and the testicular atrophy with shirinkage resulting in decreased diameter of seminiferous tubules. Pope et al., (1994) also showed reduction in the length of the testis, as a result there was a reduction in the weight of the testis. The significant decrease of the germinal epithelium may be attributed to the injury caused by AAS treatment which results in the cessation of mitosis and meiosis. Consequently, the two layers of spermatocytes reduced to a single layer thickness and a 4 – 5 layers thick zone of spermatids reduced to 2 - 3 layers and only few spermatozoa could be demonstrated in the tubular lumen. These finding are consistent with the finding by Mesbah et al., ( 2008), who suggested that this may be due to the reduction in the germ cell population and maturation or it could be due to the effect of AAS directly on the Sertoli cells which are responsible for the hormonal, nutritional, and physical support. Drugs 113 such as AASs that injure or disrupt the function of Sertoli cells can effectively educe their supportive roles and result in an increase in the elimination of the germ cell numbers via apoptosis (Skinner et al., 1985). These finding also implied that a major intratesticular change is taking place during the therapy, which accounted for the decrease in seminiferous tubule size / diameter and testicular sizes ( Heller et al., 1963 ). These findings are also confirmed by the results given by Noorafshan et al., 2005 who observed that there was reduction of the volume and the weight of the testis (P<0.01). Similar results were also given by Fainber et al., (1997) who also noted that there was reduction of the testicular weight even after withdrawing the hormonal injection for few weeks. The results of the present study showed a remarkable decrease (p<0.001) in the number and size of the interstitial cells of Leydig and depletion of intact cells in acute and chronically treated animals when compared with that of control animals. The results were consistent with those of (Ludwig, 1950; Grokett et al., 1992 and Feinberg et al., 1997); who found severe depletion of Leydig cells following treatment by AASs. The findings of Mesbah et al., (2008), also confirms our findings which showed that as a result of treatment there was less number of Leydig cells, blood vessels, and fibroblasts were observed and the interstitial space were wider. During the study, when the hormonal level of the different reproductive hormones were taken in order to evaluate the relationship between them, which showed that there was significant decreased level of these hormones 114 ( P<0.0001 ), i.e. Testosterone, Leutinizing hormone and Follicle stimulating hormones in the acute and chronic groups when compared them with the control group. As derivatives of testosterone, anabolic steroids greatly affect the male pituitary – gonadal axis. Hypogonadism can be induced, characterized decreased serum concenteration of testosterone, testicular atrophy and impaired spermatogenesis. These effects result from negative feedback of androgens from local suppression of excess androgens on the testis (Ulrich, 2002 and Saied et al., 2007). Leydig cells are known to have receptors for LH that stimulates these cells to produce testosterone (Johnson et al., 1997). Both LH and testosterone are responsible for normal spermatogenesis in male rats (Steinberger, 1971; and Zirkin, 1998). Therefore, depletion of LH receptors and decrease in peripheral LH by exogenous testosterone administration result in the reduction of testosterone secretion asreported by Bijlsma et al., 1982; Squires et al., 1982; and Ichihara et al., (2001). As the level of FSH and LH are reduced significantly (p<0.0001) in both treated groups while comparing them with control group. Spermatogenesis is under the control of FSH and LH, whose secretion is regulated by gonadal steroids (Mesbah et al., 2008). Administration of anabolic steroids, as derivatives of testosterone, suppresses gonadotropin secretion. A hypogonadal state can be induced that is characterized by decreased serum gonadotrphins, decreased serum testosterone concentration, testicular atrophy, impaired concentration and impaired spermatogenesis. 115 These effects results from the negative feedback of androgens on the hypothalamic - pituitary axis and possibly from local suppressive effect of androgens on the testis ( Kilshaw et al., 1975; Jarow and Lipshultz, 1990; Torres-Callega et al., 2001). Dohle et al., (2003) reported that exogenous administration of synthetic testosterone resulted in negative feedback on the hypothalamic-pituitary axis and Thus causes the inhibition of the secretion of both FSH and LH occurs. This explains why the spermatogenesis is affected in our study. The reason is that testicular concentration of testosterone are necessary to maintain normal length of the seminiferous tubule and the reduction in tubular length may be one reason for the reduction in the testicular weight as presented by Noorafshan et al., (2005). The present work describes the study on the reversibility of testoviron induced changes in spermatogenesis hormonal levels after discontinuing the drug in male rats. The results showed that the number and the parameters of the seminiferous tubules and the interstitial cells are returning back towards normal, although they do not return completely because following discontinuation of steroid abuse and return of normal pituitary function one would not expect the sperm concentration to start improving immediately. Once spermatogenesis is arrested it may take as long as 64 days for spermatozoa to appear in the seminiferous tubules (Helleri and Clermont, 1963). In addition, several other studies have equally shown that the negative impacts caused by the use of AASs are likely to be reversible as evident 116 from the present study. This is because testicular volume which was initially reduced following androgen in adult men (Mauss et al., 1975) normalized after discontinuation of therapy (Zachman et al., 1976; and Bra"mswig et al., 1984). CONCLUSION The desire to succeed in athletic competition can be a powerful force, driving atheltes to the illegal and potentially harmful use of AASs. The real battle and way forward is educating our youth and athletes on the danger of taking AAS The present study reveals that administration of AAS compounds have a clear effect when given for short period to see the acute changes and to see the changes when given for longer period. Administration of testoviron has an obvious effect on testicular structure including the degenerative changes in the germ cells and Leydig cells. These are accompanied by the changes seminiferous tubular parameters and testis atrophy. There was also the decline significantly in the different hormones including Testosterone, FSH and LH ( P<0.0001 ), which secondarily affects the spermatogenesis. This study suggest major adverse effects on the male reproductive organs of athletes and those who abuse AAS compounds. The use of testoviron is with caution and short intermittent therapy is desirous for better spermatogenic cycle and improved overall fertility.
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