DISCUSSION by zhangyun




          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

(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


       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.

      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

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

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

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

( 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.

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

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.,



         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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